Latest Articles Include:
- Finding the missing links in EGFR
- Nat Struct Mol Biol 19(1):1-3 (2012)
ARTICLE NAVIGATION - ISSUE Previous January 2012, Volume 19 No 1 pp1-127 * News and Views * Research Highlights * Review * Articles * Brief Communication * Technical ReportsAbout the cover News and Views Finding the missing links in EGFR - pp1 - 3 Nicholas J Bessman & Mark A Lemmon doi:10.1038/nsmb.2221 Structural studies of the epidermal growth factor receptor (EGFR) have advanced greatly in recent years, but they have used a 'divide-and-conquer' approach for independent study of the intracellular and extracellular regions. Several recent papers provide important new perspectives on 'undivided' EGFR and describe the initial steps in reconstructing signaling behavior of the intact receptor. Full Text - Finding the missing links in EGFR | PDF (638 KB) - Finding the missing links in EGFR Claims and counterclaims of X-chromosome compensation - pp3 - 5 James A Birchler doi:10.1038/nsmb.2218 Is there upregulation of the single active X chromosome in mammals or not? Recent studies take different points of view. Full Text - Claims and counterclaims of X-chromosome compensation | PDF (414 KB) - Claims and counterclaims of X-chromosome compensation See also:Article by Yildirim et al. Thresholds of replication stress signaling in cancer development and treatment - pp5 - 7 Jiri Bartek, Martin Mistrik & Jirina Bartkova doi:10.1038/nsmb.2220 Oncogene-induced replication stress and DNA damage are among the hallmarks of cancer. A recent study explores how different levels of replication stress affect animal development and tumorigenesis, and how targeting of the replication stress–signaling pathway of ATR and Chk1 kinases can be exploited for selective killing of cancer cells. Full Text - Thresholds of replication stress signaling in cancer development and treatment | PDF (8,036 KB) - Thresholds of replication stress signaling in cancer development and treatment Research Highlights * Methylating fingers * Structural basis of silencing * Stalled out * Rli1 does the splits Review New approaches for dissecting protease functions to improve probe development and drug discovery - pp9 - 16 Edgar Deu, Martijn Verdoes & Matthew Bogyo doi:10.1038/nsmb.2203 Abstract - New approaches for dissecting protease functions to improve probe development and drug discovery | Full Text - New approaches for dissecting protease functions to improve probe development and drug discovery | PDF (2,959 KB) - New approaches for dissecting protease functions to improve probe development and drug discovery | Supplementary information Articles RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks - pp17 - 24 Yoshitami Hashimoto, Fabio Puddu & Vincenzo Costanzo doi:10.1038/nsmb.2177 A system to reconstitute a collapsed replication fork using Xenopus laevis egg extracts is developed. The study shows that upon fork collapse, DNA Pol epsilon and the GINS complex are uncoupled from the replisome, and their reloading onto DNA requires repair proteins Rad51 and Mre11. Abstract - RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks | Full Text - RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks | PDF (915 KB) - RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks | Supplementary information A unique H2A histone variant occupies the transcriptional start site of active genes - pp25 - 30 Tatiana A Soboleva, Maxim Nekrasov, Anuj Pahwa, Rohan Williams, Gavin A Huttley & David J Tremethick doi:10.1038/nsmb.2161 The histone variant H2A.Bbd inhibits folding of nucleosomal arrays and reverses chromatin-mediated transcriptional repression in vitro. New studies have uncovered the related mouse H2A variant H2A.Lap1 as a novel component of the transcription start site of active genes during specific stages of spermatogenesis, which enables transcriptional activation by unfolding the chromatin locally. Abstract - A unique H2A histone variant occupies the transcriptional start site of active genes | Full Text - A unique H2A histone variant occupies the transcriptional start site of active genes | PDF (1,514 KB) - A unique H2A histone variant occupies the transcriptional start site of active genes | Supplementary information Signal-dependent dynamics of transcription factor translocation controls gene expression - pp31 - 39 Nan Hao & Erin K O'Shea doi:10.1038/nsmb.2192 The Msn2 transcription factor is translocated to the nucleus to activate transcription of hundreds of genes in response to various environmental stimuli. Experimental and computational single-molecule analyses reveal how different stimuli elicit different dynamical patterns of Msn2 translocation, which are interpreted by promoters with distinct properties to produce specific patterns of target gene expression. Abstract - Signal-dependent dynamics of transcription factor translocation controls gene expression | Full Text - Signal-dependent dynamics of transcription factor translocation controls gene expression | PDF (1,086 KB) - Signal-dependent dynamics of transcription factor translocation controls gene expression | Supplementary information Intrinsic tethering activity of endosomal Rab proteins - pp40 - 47 Sheng-Ying Lo, Christopher L Brett, Rachael L Plemel, Marissa Vignali, Stanley Fields, Tamir Gonen & Alexey J Merz doi:10.1038/nsmb.2162 Rab small G proteins regulate membrane trafficking events by recruiting effectors that mediate vesicle tethering. In vitro studies now suggest that Vps21 and other endosomal Rabs in budding yeast can undergo GTP-regulated Rab-Rab interactions that drive tethering in the absence of effectors, implying that they have an intrinsic tethering activity that may function in concert with conventional effectors. Abstract - Intrinsic tethering activity of endosomal Rab proteins | Full Text - Intrinsic tethering activity of endosomal Rab proteins | PDF (1,333 KB) - Intrinsic tethering activity of endosomal Rab proteins | Supplementary information Ndc10 is a platform for inner kinetochore assembly in budding yeast - pp48 - 55 Uhn-Soo Cho & Stephen C Harrison doi:10.1038/nsmb.2178 * PDB code * 3SQI * 3T79 * 3D view * 3SQI * 3T79 Kinetochores assemble on centromeric DNA and link centromeres to spindle microtubules, thus allowing proper segregation during mitosis. The kinetochore subunit Ndc10 makes contacts with centromeric DNA elements, which are now directly observed in a crystal structure. Along with biochemical analyses, the work indicates that Ndc10 functions as a central organizing hub to assemble the inner kinetochore. Abstract - Ndc10 is a platform for inner kinetochore assembly in budding yeast | Full Text - Ndc10 is a platform for inner kinetochore assembly in budding yeast | PDF (1,395 KB) - Ndc10 is a platform for inner kinetochore assembly in budding yeast | Supplementary information X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription - pp56 - 61 Eda Yildirim, Ruslan I Sadreyev, Stefan F Pinter & Jeannie T Lee doi:10.1038/nsmb.2195 In addition to balancing X-chromosome dosage between males and females via X inactivation, mammals also balance dosage of X chromosomes and autosomes. Allele-specific chromatin immunoprecipitation with deep sequencing (ChIP-seq) analyses now show that the active X chromosome is upregulated at the level of both transcription initiation and elongation. Abstract - X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription | Full Text - X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription | PDF (1,029 KB) - X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription | Supplementary information See also:News and Views by Birchler An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains - pp62 - 71 Julien D F Licchesi, Juliusz Mieszczanek, Tycho E T Mevissen, Trevor J Rutherford, Masato Akutsu, Satpal Virdee, Farid El Oualid, Jason W Chin, Huib Ovaa, Mariann Bienz & David Komander doi:10.1038/nsmb.2169 * PDB code * 3ZRH * 3D view * 3ZRH The OTU domain deubiquitinase TRABID specifically hydrolyzes atypical Lys29- and Lys33-linked diubiquitin chains. Structural analysis of TRABID reveals an unpredicted ankyrin-repeat domain that binds ubiquitin and is crucial for TRABID efficiency and linkage specificity in vitro and in vivo. Abstract - An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains | Full Text - An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains | PDF (2,198 KB) - An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains | Supplementary information Mechanism of mismatch recognition revealed by human MutSβ bound to unpaired DNA loops - pp72 - 78 Shikha Gupta, Martin Gellert & Wei Yang doi:10.1038/nsmb.2175 * PDB code * 3THY * 3THX * 3THW * 3THZ * 3D view * 3THY * 3THX * 3THW * 3THZ Eukaryotic MutSβ is a heterodimer composed of Msh2 and Msh3 that recognizes insertion-deletion loops (IDLs) and 3′ overhangs during mismatch repair. Now crystal structures of MutSβ in complex with DNA, containing IDLs of varying lengths, reveal that this complex interacts with its substrate differently than MutSα and bacterial MutS do. Abstract - Mechanism of mismatch recognition revealed by human MutSβ bound to unpaired DNA loops | Full Text - Mechanism of mismatch recognition revealed by human MutSβ bound to unpaired DNA loops | PDF (1,980 KB) - Mechanism of mismatch recognition revealed by human MutSβ bound to unpaired DNA loops | Supplementary information The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β1−40 peptide - pp79 - 83 Priyanka Narayan, Angel Orte, Richard W Clarke, Benedetta Bolognesi, Sharon Hook, Kristina A Ganzinger, Sarah Meehan, Mark R Wilson, Christopher M Dobson & David Klenerman doi:10.1038/nsmb.2191 Genome-wide association studies have established a link between the extracellular chaperone clusterin and susceptibility to Alzheimer's disease. A fluorescence approach is now used to reveal that clusterin sequesters Aβ1–40 oligomers and prevents them from undergoing further aggregation. Abstract - The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β1−40 peptide | Full Text - The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β1−40 peptide | PDF (625 KB) - The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β1−40 peptide | Supplementary information Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28 - pp84 - 89 Fionna E Loughlin, Luca F R Gebert, Harry Towbin, Andreas Brunschweiger, Jonathan Hall & Frdric H-T Allain doi:10.1038/nsmb.2202 * PDB code * 2LI8 * 3D view * 2LI8 Lin28 prevents the processing of pre-let-7 RNAs, but it is not clear where the Lin28 RNA binding domains interact with the RNA. The NMR structure of the Lin28 zinc knuckles with a short RNA motif reveals that each knuckle interacts with an AG dinucleotide, allowing the determination of a consensus motif for pre-let-7 recognition. Abstract - Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28 | Full Text - Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28 | PDF (1,138 KB) - Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28 | Supplementary information Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi - pp90 - 97 Caroline Thivierge, Neetha Makil, Mathieu Flamand, Jessica J Vasale, Craig C Mello, James Wohlschlegel, Darryl Conte Jr & Thomas F Duchaine doi:10.1038/nsmb.2186 The type III ribonuclease DCR-1 is essential for ERI endogenous RNAi and exogenous RNAi in Caenorhabditis elegans. A new study shows that DCR-1 forms exclusive complexes in each pathway, and characterization of the ERI complex implicates a tudor domain protein in tethering an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi. Abstract - Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi | Full Text - Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi | PDF (727 KB) - Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi | Supplementary information Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H - pp98 - 104 Ying Shen, Kyung Duk Koh, Bernard Weiss & Francesca Storici doi:10.1038/nsmb.2176 Ribonucleoside monophosphates (rNMPs) are often incorporated into genomic DNA. Misincorporated rNMPs are now shown to be repaired by mismatch repair and RNases H. If not repaired, they can serve as a template for DNA synthesis and can cause mutagenesis in Escherichia coli and yeast. Abstract - Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H | Full Text - Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H | PDF (445 KB) - Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H | Supplementary information A cis-antisense RNA acts in trans in Staphylococcus aureus to control translation of a human cytolytic peptide - pp105 - 112 Nour Sayed, Ambre Jousselin & Brice Felden doi:10.1038/nsmb.2193 Cis-encoded antisense RNAs (asRNAs) are transcribed from the DNA strand opposite another gene and function by pairing with RNAs expressed from the complementary strand. A new study provides evidence that a bacterial cis-asRNA acts in trans, using a domain outside of its target complementarity sequence, suggesting the need for a mechanistic re-evaluation of asRNA-based gene regulation. Abstract - A cis-antisense RNA acts in trans in Staphylococcus aureus to control translation of a human cytolytic peptide | Full Text - A cis-antisense RNA acts in trans in Staphylococcus aureus to control translation of a human cytolytic peptide | PDF (1,611 KB) - A cis-antisense RNA acts in trans in Staphylococcus aureus to control translation of a human cytolytic peptide | Supplementary information Brief Communication Single-molecule studies reveal the function of a third polymerase in the replisome - pp113 - 116 Roxana E Georgescu, Isabel Kurth & Mike E O'Donnell doi:10.1038/nsmb.2179 Recent work has indicated that the Escherichia coli replisome contains three DNA polymerases that are used to replicate two parental strands. A single-molecule approach is now used to compare replisomes reconstituted with two or three polymerases, revealing that the presence of a third polymerase ensures higher processivity overall and more efficient replication of the lagging strand. Abstract - Single-molecule studies reveal the function of a third polymerase in the replisome | Full Text - Single-molecule studies reveal the function of a third polymerase in the replisome | PDF (728 KB) - Single-molecule studies reveal the function of a third polymerase in the replisome | Supplementary information Technical Reports Fluorescent fusion protein knockout mediated by anti-GFP nanobody - pp117 - 121 Emmanuel Caussinus, Oguz Kanca & Markus Affolter doi:10.1038/nsmb.2180 The combination of an F-box domain with a single-domain antibody that recognizes green fluorescent protein (GFP) now allows controlled depletion of GFP fusions in mammalian cells and in flies. This system, called deGradFP, should be widely useful, as GFP fusions are available for many proteins in model organisms. Abstract - Fluorescent fusion protein knockout mediated by anti-GFP nanobody | Full Text - Fluorescent fusion protein knockout mediated by anti-GFP nanobody | PDF (1,208 KB) - Fluorescent fusion protein knockout mediated by anti-GFP nanobody | Supplementary information A metal switch for controlling the activity of molecular motor proteins - pp122 - 127 Jared C Cochran, Yu Cheng Zhao, Dean E Wilcox & F Jon Kull doi:10.1038/nsmb.2190 * PDB code * 3PXN * 3D view * 3PXN NTPases use a metal ion, typically Mg2+, coordinated by a conserved serine or threonine residue, to enable phosphate binding and catalysis. Now cysteine substitutions at the switch 1 motif of different kinesins render them able to use Mn2+ instead of Mg2+, allowing their enzymatic and motor activities to be modulated by the ratio of Mg2+ to Mn2+. Abstract - A metal switch for controlling the activity of molecular motor proteins | Full Text - A metal switch for controlling the activity of molecular motor proteins | PDF (1,145 KB) - A metal switch for controlling the activity of molecular motor proteins | Supplementary information - Claims and counterclaims of X-chromosome compensation
- Nat Struct Mol Biol 19(1):3-5 (2012)
Article preview View full access options Nature Structural & Molecular Biology | News and Views Claims and counterclaims of X-chromosome compensation * James A Birchler1Journal name:Nature Structural & Molecular BiologyVolume: 19,Pages:3–5Year published:(2012)DOI:doi:10.1038/nsmb.2218Published online 05 January 2012 Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Is there upregulation of the single active X chromosome in mammals or not? Recent studies take different points of view. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Structural & Molecular Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Affiliations * James A. Birchler is in the Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * James A Birchler Author Details * James A Birchler Contact James A Birchler Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Thresholds of replication stress signaling in cancer development and treatment
- Nat Struct Mol Biol 19(1):5-7 (2012)
Article preview View full access options Nature Structural & Molecular Biology | News and Views Thresholds of replication stress signaling in cancer development and treatment * Jiri Bartek1, 2 * Martin Mistrik2 * Jirina Bartkova1 * Affiliations * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:5–7Year published:(2012)DOI:doi:10.1038/nsmb.2220Published online 05 January 2012 Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Oncogene-induced replication stress and DNA damage are among the hallmarks of cancer. A recent study explores how different levels of replication stress affect animal development and tumorigenesis, and how targeting of the replication stress–signaling pathway of ATR and Chk1 kinases can be exploited for selective killing of cancer cells. Figures at a glance * Figure 1: Impact of different levels of ATR signaling on organismal development and tumorigenesis. Whereas wild-type and heterozygous mice develop normally, both human patients17 and the corresponding mouse model18 with pronounced genetic defects in ATR suffer from a complex developmental disorder (Seckel syndrome). The role of ATR in tumorigenesis also depends on a signaling threshold, as heterozygous mice are haploinsufficient for tumor suppression22, 23. Murga et al.4 show that excessive replication stress under in vivo conditions of very low ATR with concomitant overexpression of the Myc oncogene leads to synthetic lethality at the cellular level, resulting in exacerbation of the Seckel syndrome (*) and the virtual absence of tumors (**). * Figure 2: Distinct roles of the ATR-Chk1 pathway during multistep tumorigenesis. Oncogene activation in early lesions leads to replication stress and DNA damage, consequently triggering the DDR machinery and leading to checkpoint-imposed senescence or death of nascent tumor cells3, 4, 5, 6, 7. In tumors where the DDR barrier is overcome (for example, by selection for p53 mutations), disease progression may ensue. The advanced tumors still experience the oncogene-induced replication stress and genetic instability and often adapt to such challenge. In the context of disabled cell-cycle checkpoints and apoptosis, the abilities of the ATR-Chk1 signaling module to support the replication machinery and promote DNA repair can thus be 'hijacked' to boost the overall fitness of the malignant tumor. * Figure 3: Potential exploitation of replication stress as a target for cancer therapy. Many, but not all, tumors feature enhanced replication stress (RS)4, 12, 24. Subsets of cancers of diverse tissue origin might therefore be examined for markers of replication stress and/or activated RSR in order to select individuals who might benefit from treatment with drugs that inhibit the ATR or Chk1 kinases. Examples of preclinical results of such a treatment approach are reported by Murga et al.4. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Structural & Molecular Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Affiliations * Jiri Bartek and Jirina Bartkova are at the Centre for Genotoxic Stress Research, Danish Cancer Society, Copenhagen, Denmark. * Martin Mistrik and Jiri Bartek are at the Institute of Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jiri Bartek Author Details * Jiri Bartek Contact Jiri Bartek Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Mistrik Search for this author in: * NPG journals * PubMed * Google Scholar * Jirina Bartkova Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - New approaches for dissecting protease functions to improve probe development and drug discovery
- Nat Struct Mol Biol 19(1):9-16 (2012)
Nature Structural & Molecular Biology | Review New approaches for dissecting protease functions to improve probe development and drug discovery * Edgar Deu1 * Martijn Verdoes1 * Matthew Bogyo1, 2 * Affiliations * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:9–16Year published:(2012)DOI:doi:10.1038/nsmb.2203Published online 05 January 2012 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Proteases are well-established targets for pharmaceutical development because of their known enzymatic mechanism and their regulatory roles in many pathologies. However, many potent clinical lead compounds have been unsuccessful either because of a lack of specificity or because of our limited understanding of the biological roles of the targeted protease. In order to successfully develop protease inhibitors as drugs, it is necessary to understand protease functions and to expand the platform of inhibitor development beyond active site–directed design and in vitro optimization. Several newly developed technologies will enhance assessment of drug selectivity in living cells and animal models, allowing researchers to focus on compounds with high specificity and minimal side effects in vivo. In this review, we highlight advances in the development of chemical probes, proteomic methods and screening tools that we feel will help facilitate this paradigm shift in drug discovery. View full text Figures at a glance * Figure 1: Mechanism of substrate hydrolysis by the primary families of proteases. () Protease substrates bind through interactions of the side chain residues (P and P′ residues) with the substrate pockets of the protease (S and S′ pockets). The red dashed line indicates a scissile bond. (–) The architecture of the active site and mechanism of hydrolysis for N-terminal threonine, serine and cysteine proteases that use an acyl-enzyme intermediate formed through nucelophilic attack by the catalytic side chain residue. (,) In the case of zinc metalloproteases (), aspartate proteases () and glutamate proteases (not depicted), a carboxylic acid group or metal ion activates a water molecule, leading to acid-base catalysis. The seventh and newest protease family, the asparagine peptide lyases, cleave themselves using an asparagine residue as the nucleophile2 (not depicted). * Figure 2: Schematic presentation of the hit-to-lead process. () In a classical protease drug discovery approach, the emphasis of screening and optimization is on maximizing the potency of a hit compound for a recombinant protease. Off-target effects and efficacy are usually tested after the optimization process, and problems encountered when testing the compounds in cultures and in vivo require either modifying the structure of the lead inhibitor to solve a particular issue or selecting a different chemotype for further optimization. () In this review, we propose a holistic approach, in which the emphasis is on identifying hits in a more complex and relevant context (intact cells), incorporating the specificity profile of hits to identify and optimize lead compounds. We believe that placing the emphasis of the hit-to-lead optimization process on selectivity instead of just on potency will help prevent off-target effects and thus increase the chances for developing protease inhibitor drugs with minimum side effects. * Figure 3: Activity-based probes report on tightly regulated protease activity. () Proteases are not only regulated on the transcription and translation levels but also highly regulated on the protein level. Expressed as zymogens, proteases are activated in a variety of ways and by a variety of factors, depending on the protease, including allosteric activation, environmental changes, localization, protein-protein interactions and processing by upstream proteases. Endogenous inhibitors and targeted degradation form yet another layer of regulation. () Activity-based probes are small-molecule reporters that use the active protease's own chemistry to distinguish it from its zymogen or inhibited form. Most ABPs consist of three parts: a warhead (an electrophilic moiety that reacts with the active site nucleophile to result in a covalent and irreversible adduct), a spacer and/or recognition element that targets the probe to a specific target protease and a tag (usually a fluorescent dye and/or an affinity handle, like biotin). * Figure 4: Chemical tools to study protease function and to measure target inhibition. () Forward chemical genetics allows for target identification through the introduction of an affinity tag to the hit compound. () Near-infrared fluorescently labeled ABPs can be applied to top-down characterization of a target protease. Whole-animal noninvasive imaging techniques allow the visualization of target distribution, and extracted tissue can be analyzed ex vivo. Histology shows target distribution on a microscopic level, FACS analysis identifies the types of cells that contain active protease and biochemical analysis allows characterization at the protein level. Treatment with a lead compound before labeling provides information on target inhibition. Mouse images are from our previous publication39. () Broad-spectrum protease probes enable a readout of the inhibition profile of a lead compound for an entire protease family in a proteome. Members of the targeted family can be resolved on a gel, and inhibition results in diminished labeling of individual proteases. (! ) Global profiling of all reactive cysteines in a proteome; iodoacetamide-based reporter molecules will react primarily with the more reactive cysteines. Using isotopically labeled reporter molecules, this method can be used to predict functional cysteines in proteomes as well as to identify targets. When the methods described here are used to evaluate the specificity profile of a reversible inhibitor, the labeling conditions should be adjusted so that the covalent probe does not outcompete the inhibitor. Because these methods have a good dynamic range, this can be accomplished by lowering the probe concentration or decreasing the labeling times. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Pathology, Stanford University School of Medicine, Stanford, California, USA. * Edgar Deu, * Martijn Verdoes & * Matthew Bogyo * Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA. * Matthew Bogyo Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Matthew Bogyo Author Details * Edgar Deu Search for this author in: * NPG journals * PubMed * Google Scholar * Martijn Verdoes Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew Bogyo Contact Matthew Bogyo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (74K) Supplementary Box 1 Additional data - RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks
- Nat Struct Mol Biol 19(1):17-24 (2012)
Nature Structural & Molecular Biology | Review New approaches for dissecting protease functions to improve probe development and drug discovery * Edgar Deu1 * Martijn Verdoes1 * Matthew Bogyo1, 2 * Affiliations * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:9–16Year published:(2012)DOI:doi:10.1038/nsmb.2203Published online 05 January 2012 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Proteases are well-established targets for pharmaceutical development because of their known enzymatic mechanism and their regulatory roles in many pathologies. However, many potent clinical lead compounds have been unsuccessful either because of a lack of specificity or because of our limited understanding of the biological roles of the targeted protease. In order to successfully develop protease inhibitors as drugs, it is necessary to understand protease functions and to expand the platform of inhibitor development beyond active site–directed design and in vitro optimization. Several newly developed technologies will enhance assessment of drug selectivity in living cells and animal models, allowing researchers to focus on compounds with high specificity and minimal side effects in vivo. In this review, we highlight advances in the development of chemical probes, proteomic methods and screening tools that we feel will help facilitate this paradigm shift in drug discovery. View full text Figures at a glance * Figure 1: Mechanism of substrate hydrolysis by the primary families of proteases. () Protease substrates bind through interactions of the side chain residues (P and P′ residues) with the substrate pockets of the protease (S and S′ pockets). The red dashed line indicates a scissile bond. (–) The architecture of the active site and mechanism of hydrolysis for N-terminal threonine, serine and cysteine proteases that use an acyl-enzyme intermediate formed through nucelophilic attack by the catalytic side chain residue. (,) In the case of zinc metalloproteases (), aspartate proteases () and glutamate proteases (not depicted), a carboxylic acid group or metal ion activates a water molecule, leading to acid-base catalysis. The seventh and newest protease family, the asparagine peptide lyases, cleave themselves using an asparagine residue as the nucleophile2 (not depicted). * Figure 2: Schematic presentation of the hit-to-lead process. () In a classical protease drug discovery approach, the emphasis of screening and optimization is on maximizing the potency of a hit compound for a recombinant protease. Off-target effects and efficacy are usually tested after the optimization process, and problems encountered when testing the compounds in cultures and in vivo require either modifying the structure of the lead inhibitor to solve a particular issue or selecting a different chemotype for further optimization. () In this review, we propose a holistic approach, in which the emphasis is on identifying hits in a more complex and relevant context (intact cells), incorporating the specificity profile of hits to identify and optimize lead compounds. We believe that placing the emphasis of the hit-to-lead optimization process on selectivity instead of just on potency will help prevent off-target effects and thus increase the chances for developing protease inhibitor drugs with minimum side effects. * Figure 3: Activity-based probes report on tightly regulated protease activity. () Proteases are not only regulated on the transcription and translation levels but also highly regulated on the protein level. Expressed as zymogens, proteases are activated in a variety of ways and by a variety of factors, depending on the protease, including allosteric activation, environmental changes, localization, protein-protein interactions and processing by upstream proteases. Endogenous inhibitors and targeted degradation form yet another layer of regulation. () Activity-based probes are small-molecule reporters that use the active protease's own chemistry to distinguish it from its zymogen or inhibited form. Most ABPs consist of three parts: a warhead (an electrophilic moiety that reacts with the active site nucleophile to result in a covalent and irreversible adduct), a spacer and/or recognition element that targets the probe to a specific target protease and a tag (usually a fluorescent dye and/or an affinity handle, like biotin). * Figure 4: Chemical tools to study protease function and to measure target inhibition. () Forward chemical genetics allows for target identification through the introduction of an affinity tag to the hit compound. () Near-infrared fluorescently labeled ABPs can be applied to top-down characterization of a target protease. Whole-animal noninvasive imaging techniques allow the visualization of target distribution, and extracted tissue can be analyzed ex vivo. Histology shows target distribution on a microscopic level, FACS analysis identifies the types of cells that contain active protease and biochemical analysis allows characterization at the protein level. Treatment with a lead compound before labeling provides information on target inhibition. Mouse images are from our previous publication39. () Broad-spectrum protease probes enable a readout of the inhibition profile of a lead compound for an entire protease family in a proteome. Members of the targeted family can be resolved on a gel, and inhibition results in diminished labeling of individual proteases. (! ) Global profiling of all reactive cysteines in a proteome; iodoacetamide-based reporter molecules will react primarily with the more reactive cysteines. Using isotopically labeled reporter molecules, this method can be used to predict functional cysteines in proteomes as well as to identify targets. When the methods described here are used to evaluate the specificity profile of a reversible inhibitor, the labeling conditions should be adjusted so that the covalent probe does not outcompete the inhibitor. Because these methods have a good dynamic range, this can be accomplished by lowering the probe concentration or decreasing the labeling times. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Pathology, Stanford University School of Medicine, Stanford, California, USA. * Edgar Deu, * Martijn Verdoes & * Matthew Bogyo * Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA. * Matthew Bogyo Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Matthew Bogyo Author Details * Edgar Deu Search for this author in: * NPG journals * PubMed * Google Scholar * Martijn Verdoes Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew Bogyo Contact Matthew Bogyo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (74K) Supplementary Box 1 Additional data - A unique H2A histone variant occupies the transcriptional start site of active genes
- Nat Struct Mol Biol 19(1):25-30 (2012)
Nature Structural & Molecular Biology | Article RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks * Yoshitami Hashimoto1 * Fabio Puddu1 * Vincenzo Costanzo1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:17–24Year published:(2012)DOI:doi:10.1038/nsmb.2177Received 16 May 2011 Accepted 29 September 2011 Published online 04 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In higher eukaryotes, the dynamics of replisome components during fork collapse and restart are poorly understood. Here we have reconstituted replication fork collapse and restart by inducing single-strand DNA lesions that create a double-strand break in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45–MCM–GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and polymerase ɛ onto DNA in a fashion that is dependent on RAD51 and MRE11 but independent of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process. View full text Figures at a glance * Figure 1: RAD51 is required for DNA replication in the presence of forks collapsed by a single-strand break in the template. () The requirement of RAD51 and PCNA modification at Lys164 for replication of undamaged sperm DNA (control) or MMS- or UV-treated sperm DNA was tested using GST-labeled BRC4, which sequesters RAD51, and PCNA K164R mutant. Replication products (labeled with [32P]dATP) were resolved by neutral agarose gel electrophoresis and subjected to autoradiography (left). The quantification of the signal is shown in the graph as photon emission intensity expressed in arbitrary units (AU) (right). The data shown here and hereafter represent typical findings of three or more experiments. () A model for ssDNA-specific endonuclease-dependent fork collapse and RAD51-dependent restart. () The requirement of RAD51 for DNA replication in the presence of S1 nuclease was tested using sperm nuclei (4,000 nuclei per microliter) incubated for 80 min in the presence or absence of 1 μg ml−1 aphidicolin (Low aph) and S1 nuclease (0, 2.92, 1.46, 0.73, 0.37 U μl−1). Replication products were detect! ed by autoradiography with quantification shown on the right, as in . SYBR Green staining shows total DNA. * Figure 2: RAD51 is required for stable chromatin association of fork proteins in the presence of template breakage. () Association of fork proteins to chromatin isolated from extracts treated with GST or GST-BRC4 and 0, 2.92, 0.97 and 0.32 U μl−1 S1 nuclease in the presence of 1 μg ml−1 aphidicolin (Low aph). () Chromatin status of fork proteins, histone H2AX and PCNA in extracts treated with GST or GST-BRC4 2.92 (1/100) and 0.37 (1/800) U μl−1 S1 nuclease and aphidicolin. () Chromatin binding of PSF2 and CDC45 in the presence of 0.97 U μl−1 S1 nuclease in mock- or RAD51-depleted (–RAD51) extracts. () Chromatin binding of the indicated proteins over time in extracts treated with GST or GST-BRC4 and 1.46 U μl−1 of S1 nuclease and aphidicolin. () Nuclear CHK1 phosphorylation (P-CHK1) on Ser345 in extracts treated with 1 μg ml−1 aphidicolin alone or in combination with 1.46 U μl−1 of S1 nuclease. In –, western blotting was performed using antibodies against the indicated chromatin binding factors. 'Ext' in and indicates lanes containing 0.5 μl egg extract loaded as ! a control. Chromatin and nuclear fractions were isolated 60 min after the addition of sperm DNA to egg extracts unless otherwise indicated. * Figure 3: RAD51 is required for origin-independent fork restart and reloading of replisome components after fork collapse. (,) Replication fork restart was monitored following incubation of sperm nuclei in the first extract for 60 min with or without 10 μg ml−1 aphidicolin, and then nuclear fractions that were untreated or briefly incubated with mung bean nuclease were transferred to a second extract containing 320 nM geminin, 1 mM roscovitine and GST or GST-BRC4 () or to mock- or RAD51-depleted (–RAD51) extracts containing recombinant RAD51 (rRAD51) (). Replication products were monitored by incorporation of [32P]dATP added to the second extract and resolved by alkaline () or neutral () agarose gel electrophoresis followed by autoradiography. Quantification of signals is shown at the bottom of the gel in and in the graph in . () Chromatin binding of RAD51 and CDC45 was monitored in egg extracts that were mock- or RAD51-depleted and supplemented with the indicated amount of rRAD51. () The status of replication fork proteins bound to chromatin isolated from extracts treated as in . * Figure 4: MRE11 nuclease activity is required for DNA replication upon fork collapse. (,) Effects of the MRE11 nuclease inhibitor mirin on replication of sperm nuclei that were untreated or treated with MMS in the presence or absence of GST-BRC4 () or on sperm nuclei incubated in extracts treated with 0, 0.73, 0.37 or 0.18 U μl−1 S1 nuclease and aphidicolin (). Replication products were monitored by [32P]dATP incorporation and resolved on neutral agarose gels, which were subjected to autoradiography. Signal intensities are reported in the graphs. () Effect of mirin on replication proteins bound to chromatin isolated after a 50-min incubation in extracts treated with 0, 1.46, 0.73, 0.37 or 0.18 U μl−1 S1 nuclease. () Binding of the indicated fork proteins to chromatin incubated for 45 min in egg extracts that were untreated or supplemented with 0.73 U μl−1 S1 nuclease and mirin following protein cross-linking, sonication-induced DNA fragmentation and immunoprecipitation with control and anti-CDC45 serum. 'Ext' indicates 0.5 μl egg extract loaded as a! control in and . * Figure 5: The role of PCNA in DNA replication and chromatin association of replication proteins upon fork collapse. () Replication of sperm nuclei incubated in extracts for 80 min in the presence of 1 μg ml−1 aphidicolin and 0, 0.73, 0.37 or 0.18 U μl−1 S1 nuclease and wild-type PCNA (WT), PCNA K164R (KR), PCNA Y249A Y250A (YA) or PCNA K164R Y249A Y250A (KR YA) recombinant proteins. Replication products were resolved by neutral agarose gel electrophoresis and subjected to autoradiography (left). Signal intensities are shown in the graph (right). () Binding to chromatin of the indicated proteins was monitored by immunoblotting of chromatin treated with 200 J m−2 UV or incubated in extracts treated with 1 μg ml−1 aphidicolin, 0.97 U μl−1 S1 nuclease or EcoRI and recombinant wild-type PCNA, PCNA K164R or PCNA Y249A Y250A as indicated. 'Ext' indicates 0.5 μl egg extract loaded as a control. () The interaction of PCNA and replication proteins in egg extract was monitored by incubation of His6-tagged wild type and mutant PCNA proteins followed by pull-down with Ni-NTA–Sepharose! . The interacting proteins were detected by immunoblotting as indicated. * Figure 6: A model of replication fork collapse and restart. The presence of a ssDNA lesion in the template creates a one-sided DSB upon passage of the replisome (1), leading to the dissociation of the GINS and Pol ɛ from the fork, whereas MCM and CDC45 remain stably bound to collapsed fork (2). The one-sided DSB undergoes MRE11-mediated nuclease resection and RAD51-dependent strand annealing and invasion of the intact template. The MRE11 complex might also tether the broken DNA strand to the intact one (3). This process requires BIR-proficient PCNA, which promotes Pol η–dependent strand extension (4). Reloading of the GINS and Pol ɛ in an origin-independent fashion promotes reassembly of a functional replisome (5). Author information * Abstract * Author information * Supplementary information Affiliations * Genome Stability Unit, Clare Hall Laboratories, London Research Institute, South Mimms, Hertfordshire, UK. * Yoshitami Hashimoto, * Fabio Puddu & * Vincenzo Costanzo Contributions Y.H. and F.P. performed experiments. Y.H. and V.C. analyzed the results and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Vincenzo Costanzo Author Details * Yoshitami Hashimoto Search for this author in: * NPG journals * PubMed * Google Scholar * Fabio Puddu Search for this author in: * NPG journals * PubMed * Google Scholar * Vincenzo Costanzo Contact Vincenzo Costanzo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3.5 MB) Supplementary Figures 1–6 Additional data Entities in this article * DNA repair protein RAD51 homolog A rad51-a Xenopus laevis * View in UniProt * View in Entrez Gene * Double-strand break repair protein MRE11 mre11a Xenopus laevis * View in UniProt * View in Entrez Gene * Cell division control protein 45 homolog cdc45 Xenopus laevis * View in UniProt * View in Entrez Gene * Proliferating cell nuclear antigen pcna Xenopus laevis * View in UniProt * View in Entrez Gene * Proliferating cell nuclear antigen PCNA Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * E3 ubiquitin-protein ligase RAD18 RAD18 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Protein RecA recA Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA repair protein RAD51 homolog 1 RAD51 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Double-strand break repair protein MRE11A MRE11A Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * DNA repair protein RAD50 RAD50 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Nibrin NBN Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * DNA polymerase delta subunit 3 POL32 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Origin recognition complex subunit 1 ORC1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Origin recognition complex subunit 6 ORC6 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Cell division control protein 6 CDC6 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Cell division cycle protein CDT1 TAH11 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA replication licensing factor MCM2 MCM2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA replication licensing factor MCM7 MCM7 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Cell division control protein 45 CDC45 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA replication complex GINS protein SLD5 SLD5 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA replication complex GINS protein PSF1 PSF1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA replication complex GINS protein PSF2 PSF2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA replication complex GINS protein PSF3 PSF3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Breast cancer type 2 susceptibility protein BRCA2 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Geminin GMNN Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * GINS complex subunit 4 (Sld5 homolog) gins4 Xenopus laevis * View in UniProt * View in Entrez Gene * DNA replication complex GINS protein PSF2 gins2 Xenopus laevis * View in UniProt * View in Entrez Gene * DNA replication licensing factor mcm2 mcm2 Xenopus laevis * View in UniProt * View in Entrez Gene * Histone H2A type 1 h2afx Xenopus laevis * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase Chk1 chek1 Xenopus laevis * View in UniProt * View in Entrez Gene * DNA replication licensing factor mcm7-A mcm7-a Xenopus laevis * View in UniProt * View in Entrez Gene * Proliferating cell nuclear antigen POL30 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ubiquitin-like-specific protease 1 ULP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Maternal DNA replication licensing factor mcm3 mcm3 Xenopus laevis * View in UniProt * View in Entrez Gene * DNA replication licensing factor mcm5-A mcm5-a Xenopus laevis * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase Chk1 CHEK1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Origin recognition complex subunit 2 orc2 Xenopus laevis * View in UniProt * View in Entrez Gene - Signal-dependent dynamics of transcription factor translocation controls gene expression
- Nat Struct Mol Biol 19(1):31-39 (2012)
Nature Structural & Molecular Biology | Article A unique H2A histone variant occupies the transcriptional start site of active genes * Tatiana A Soboleva1 * Maxim Nekrasov1 * Anuj Pahwa1 * Rohan Williams1, 2 * Gavin A Huttley1 * David J Tremethick1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:25–30Year published:(2012)DOI:doi:10.1038/nsmb.2161Received 02 June 2011 Accepted 21 September 2011 Published online 04 December 2011 Corrected online11 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Transcriptional activation is controlled by chromatin, which needs to be unfolded and remodeled to ensure access to the transcription start site (TSS). However, the mechanisms that yield such an 'open' chromatin structure, and how these processes are coordinately regulated during differentiation, are poorly understood. We identify the mouse (Mus musculus) H2A histone variant H2A.Lap1 as a previously undescribed component of the TSS of active genes expressed during specific stages of spermatogenesis. This unique chromatin landscape also includes a second histone variant, H2A.Z. In the later stages of round spermatid development, H2A.Lap1 dynamically loads onto the inactive X chromosome, enabling the transcriptional activation of previously repressed genes. Mechanistically, we show that H2A.Lap1 imparts unique unfolding properties to chromatin. We therefore propose that H2A.Lap1 coordinately regulates gene expression by directly opening the chromatin structure of the TSS at ge! nes regulated during spermatogenesis. View full text Figures at a glance * Figure 1: H2A.Lap1 dynamically loads onto the sex chromosomes in late round spermatids. () Seminiferous tubule sections were indirectly immunostained with Lap1 antibodies (red) and stained with peanut agglutinin Alexa Fluor 488 conjugate (LectinPNA, green) to allow identification of the various spermatid stages on the basis of acrosome maturation9. DNA was costained with DAPI (blue). P, mid-pachytene spermatocytes; LP, late-pachytene spermatocytes; RS, round spermatids. Arrow marks residual bodies (RB; Supplementary Fig. 8). Scale bars, 25 μm. () Surface spreads of leptotene and zygotene cells were prepared from pre-pachytene testes of 12-day-old mice. Surface spreads of pachytene spermatocytes, round spermatids and elongating spermatids were prepared from adult mice. Different cell types were immunostained with SCP3 (a marker for the progression of chromosome synapsis), γX (a component of the XY body in pachytene spermatocytes) or macroH2A1.2 (enriched in centromeric heterochromatin of the Y chromosome in round spermatids). Filled white arrow, XY body. Unfil! led white arrow, Y chromosome centromeric heterochromatin. Scale bar, 10 μm. () Fluorescence in situ hybridization analyses of round spermatids using specific chromosome-X or chromosome-Y paints (reproduced with permission from our previous published study5). White lines in merged images indicate the paths used to determine fluorescence intensity across the sex chromosomes and the chromocenter, graphed at right. C, hromocenter. Scale bars, 10 μm. () Round spermatids were immunostained with Lap1 antibodies and stained with DAPI. Round spermatids were also stained with LectinPNA to distinguish whether a round spermatid was at an early or late stage of development. White lines in merged images indicate the paths used to determine the fluorescence intensity of Lap1 across the DAPI stained sex chromosome, graphed at right. Scale bar, 10 μm. * Figure 2: H2A.Lap1 is located at the TSS of active genes. () H2A.Lap1 ChIP profiles on genes active in round-spermatid X-chromosomes. Each line represents 50 genes, grouped by expression level using published gene expression data6; coloring indicates average gene expression rank in the group. The sum of frequency tag counts in the group is plotted at each base pair relative to the TSS. () Lap1 ChIP profile showing sum of frequency tag counts on 44 X-linked Group C genes, aligned between −1 kb and +1 kb from the TSS. (,) Lap1 ChIP profiles for genes on chromosome 1 () and for the whole genome (), grouped by expression level using global expression of all mouse genes in the 30-day-old testis. We separated 23 groups of 50 genes on chromosome 1 and 201 groups of 100 genes in the whole genome; coloring is as in . Note that for the whole-genome plot, the sum of all shown frequency tag counts equals 1. () Lap1 ChIP profile for the 1,000 most highly expressed mouse genes in the 30-day-old testis, aligned between −5 and +5 kb from the T! SS. () Lap1 ChIP profiles for all mouse genes expressed at the pachytene stage, grouped by expression level (164 groups of 100 genes) using published pachytene expression data6. Coloring is as in . * Figure 3: Targeting of H2A.Lap1 to X chromosome–linked genes occurs in late round spermatids. Six round spermatid–specific X-linked genes were chosen for H2A.Lap1 ChIP and gene expression analyses. () H2A.Lap1 enrichment for each gene in mouse testes at 18, 24, or 30 d of development, relative to Dusp21 at 18 d. H2A.Lap1 signal was assayed by ChIP. ChIP-seq libraries, normalized to the same DNA concentration, were analyzed by quantitative PCR using gene-specific primers that target the TSS. Data shown are means and s.d. of three repeats. () The mRNA level of each gene at each stage of testes development, determined by real-time quantitative PCR, relative to β-actin. Data shown are means and s.d. of three repeats. () H2A.Z ChIP profiles at TSS of all mouse genes expressed in the 30-day-old testis; genes are grouped by average gene expression rank as in Figure 2d (201 groups of 100 genes). () Normalized ChIP profiles of H2A.Z and H2A.Lap1 (with confidence intervals estimated by resampling) for the 1,000 most highly expressed genes in the 30-day-old mouse testis. () ! Cartoon depicting the location of H2A.Z- and H2A.Lap1-containing nucleosomes at the −2 and −1 positions, respectively, relative to the TSS. * Figure 4: H2A.Lap1 has gained a single acidic amino acid residue, which enables nucleosome arrays to partially fold. () Sedimentation coefficient distribution plots of arrays containing human Bbd in the absence or presence of 0.3 mM MgCl2 (reproduced from our previous published study3). (,) Sedimentation coefficient distribution plots of arrays containing wild-type (WT) H2A or Lap1 in the absence or presence of 1.2 mM MgCl2. (,) Sedimentation coefficient distribution plots of arrays containing WT H2A, Lap1 or mutant Lap1 (D100T) in the absence or presence of 0.4 mM MgCl2. Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE29781 Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationCorrected online 11 December 2011In the version of this article initially published, the incorrect PDB code for the MthK open channel structure was provided in the legend to Figure 1. The correct PDB code for this structure is 1LNQ. The error has been corrected in the HTML and PDF versions of the article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Affiliations * The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia. * Tatiana A Soboleva, * Maxim Nekrasov, * Anuj Pahwa, * Rohan Williams, * Gavin A Huttley & * David J Tremethick * Present address: Singapore Centre on Environmental Life Sciences Engineering, National University of Singapore, Singapore. * Rohan Williams Contributions T.A.S. helped design the experiments, cloned H2A.Lap, conducted all spermatogenesis experiments, prepared chromatin for ChIP-seq experiments and conducted the gene expression and ChIP experiments on individual X-chromosome genes. M.N. conducted the biochemical and biophysical experiments on the nucleosome arrays and prepared DNA ChIP libraries for high-throughput sequencing. R.W. developed and did data analysis of global mouse gene expression data. G.A.H. designed and executed the analysis of the Illumina short-read data. A.P. assisted with the analyses of Illumina short-read data. D.J.T. conceived the project, helped design the experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David J Tremethick Author Details * Tatiana A Soboleva Search for this author in: * NPG journals * PubMed * Google Scholar * Maxim Nekrasov Search for this author in: * NPG journals * PubMed * Google Scholar * Anuj Pahwa Search for this author in: * NPG journals * PubMed * Google Scholar * Rohan Williams Search for this author in: * NPG journals * PubMed * Google Scholar * Gavin A Huttley Search for this author in: * NPG journals * PubMed * Google Scholar * David J Tremethick Contact David J Tremethick Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * Histone H2A.Z H2afz Mus musculus * View in UniProt * View in Entrez Gene * Histone H2A.x H2afx Mus musculus * View in UniProt * View in Entrez Gene * Core histone macro-H2A.1 H2afy Mus musculus * View in UniProt * View in Entrez Gene * Dual specificity phosphatase 21 Dusp21 Mus musculus * View in UniProt * View in Entrez Gene * MCG52127 4930557A04Rik Mus musculus * View in UniProt * View in Entrez Gene - Intrinsic tethering activity of endosomal Rab proteins
- Nat Struct Mol Biol 19(1):40-47 (2012)
Nature Structural & Molecular Biology | Article Signal-dependent dynamics of transcription factor translocation controls gene expression * Nan Hao1, 2, 3 * Erin K O'Shea1, 2, 3, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:31–39Year published:(2012)DOI:doi:10.1038/nsmb.2192Received 13 June 2011 Accepted 13 October 2011 Published online 18 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Information about environmental stimuli is often transmitted using common signaling molecules, but the mechanisms that ensure signaling specificity are not entirely known. Here we show that the identities and intensities of different stresses are transmitted by modulation of the amplitude, duration or frequency of nuclear translocation of the Saccharomyces cerevisiae general stress response transcription factor Msn2. Through artificial control of the dynamics of Msn2 translocation, we reveal how distinct dynamical schemes differentially affect reporter gene expression. Using a simple model, we predict stress-induced reporter gene expression from single-cell translocation dynamics. We then demonstrate that the response of natural target genes to dynamical modulation of Msn2 translocation is influenced by differences in the kinetics of promoter transitions and transcription factor binding properties. Thus, multiple environmental signals can trigger qualitatively different dyna! mics of a single transcription factor and influence gene expression patterns. View full text Figures at a glance * Figure 1: Msn2 translocates to the nucleus with different dynamics in response to different stresses. (–) Time traces of YFP-tagged Msn2 nuclear translocation are shown for glucose limitation (), osmotic stress () and oxidative stress (). In each panel, top row: averages of single-cell time traces of Msn2-YFP translocation in response to the indicated stresses (solid circles, averages of single-cell experimental data; solid lines, s.d. of single-cell responses of ~60 cells, from at least two independent experiments); bottom row: representative single-cell time traces of Msn2-YFP nuclear translocation. AU, arbitrary units of fluorescence. Additional single-cell traces are shown in Supplementary Figure 1a,b. * Figure 2: Quantification of single-cell Msn2-YFP translocation traces. () A schematic defines the initial peak of Msn2 nuclear translocation and subsequent sporadic bursts in a single-cell time trace. () Duration (top row) and amplitude (middle row) of the initial peak are quantified for the indicated stress conditions (open circles, mean value of single cells; error bars, s.d. of single-cell responses of ~60 cells, from at least two independent experiments). Duration is not quantified for the H2O2 treatment, because a sustained translocation event was observed under this condition. () Frequency, amplitude, burst duration and interval durations of sporadic bursts in response to glucose limitation. Frequency of sporadic bursts under osmotic stress is also quantified (right). The distributions of amplitude, duration, and frequency of sporadic nuclear burst in response to glucose limitation are shown in Supplementary Figure 2a–c. Autocorrelation analysis of Msn2 localization traces upon glucose limitation are presented in Supplementary Figure 2d. * Figure 3: Experimental and computational analysis of gene expression in response to modulation of Msn2 nuclear translocation dynamics. () A diagram describes the analog-sensitive system used to control Msn2 nuclear translocation. () Gene expression model. A detailed description of the model construction and fitting procedure are included in Supplementary Results. () Averages of single-cell time traces of Msn2-YFP nuclear localization and reporter gene expression (cyan fluorescent protein, CFP) measured in the same cells in response to inhibitor treatments (black solid circles, averages of time-trace data; black solid lines, s.d. of single-cell data of ~50 cells, from at least two independent experiments; green solid line, curve fitting of Msn2 translocation traces; red solid line, model simulation). The time traces of Msn2 nuclear localization were fit with a piecewise exponential function (Supplementary Fig. 3) to produce continuous time-dependent profiles, TF(t), which served as input for the model. The model in was fit to the averages of single-cell time traces of reporter gene expression (Supplementary ! Results). The complete dataset is included in Supplementary Figures 3–5. * Figure 4: The dynamics of Msn2 nuclear translocation influences target gene expression. () The relationship between gene expression and the area under the curve (AUC) of Msn2 inputs (open circles, experimental data; solid lines, model simulation; error bars, s.d. of single-cell data of ~50 cells, from at least two independent experiments). The integrals of Msn2 inputs were quantified from the data in Supplementary Figures 3–5. Single Msn2 inputs with 10 min (blue), 20 min (red) or 40 min (black) durations were compared with oscillatory Msn2 inputs for 5-min pulse duration (orange). () Relationship between dynamic of Msn2 nuclear inputs and reporter gene expression. Left, gene expression versus Msn2 input amplitude (input duration: black = 40 min; red = 20 min; blue = 10 min); center, gene expression versus Msn2 input duration (input amplitude: yellow green = 2,190 AU; orange = 1,751 AU; black = 1,309 AU; green = 1,010 AU; red = 672 AU; blue = 406 AU); right, gene expression versus Msn2 input frequency (input amplitude = 1,751 AU; pulse duration = 5 min). () M! odel simulations reproduce the measured expression responses to natural stresses. For the indicated stress conditions, each single-cell trace of Msn2 translocation was used as input for the gene expression model. The simulated single-cell expression traces were averaged to generate the simulation curves (solid lines, top row) and compared with averages of measured single-cell expression (solid circles, bottom row). * Figure 5: The model predicts that target genes have distinct responses to different input regimes. Two sets of parameters were varied, and alterations in gene expression output were predicted using the expression model: parameters that govern transcription factor binding, Kd and n; and parameters that govern kinetics of promoter transition, k1 and k2. The inputs are selected to be in the physiological ranges of the natural stress responses (Fig. 2). () The expression curves upon amplitude modulation (AM), duration modulation (DM) and frequency modulation (FM) (left column) and the expression ratios (the ratio of gene expression level upon low stimulus to expression level upon high stimulus) calculated from the expression curves (right column, same genes use same colors for curves and ratios) are shown for hypothetical genes with different binding parameters (Kd, n) and the same promoter kinetics (k1, k2). The values below the bar graph represent the fold changes from the parameter values obtained from fitting the reporter response data (Fig. 3). () Model predictions are s! hown for hypothetical genes with the same binding parameters and different promoter kinetics. () Natural target genes may differ in both binding parameters and promoter kinetics. Model predictions for four hypothetical genes with different binding parameters and different promoter kinetics. Genes 1 and 2 have the same slow promoter kinetics, whereas Genes 3 and 4 have the same fast promoter kinetics. Genes 1 and 3 have the same high transcription factor binding, whereas Genes 2 and 4 have the same low transcription factor binding. * Figure 6: Analysis of a simplified model. () Schematic of the simplified model. () The model behaviors in response to duration modulation inputs when the timescale of promoter transition is longer () and shorter () than input duration. The input durations are Ta and Tb. () The model behaviors in response to frequency modulation inputs. () The responses when the timescales of promoter activation and deactivation are longer than pulse duration and pulse interval, respectively. (–) The responses when the timescale of promoter activation is shorter than pulse duration or when the timescale of promoter inactivation is shorter than pulse interval. Pulse duration is Ton; the interval durations are Toff_a and Toff_b. For and , ω, inputs; P2, promoter activity; R, gene product. () The simulated relationship between gene expression output and input duration. Equations used in simulations are indicated; these relationships are calculated with the median value of the input duration we used in the simulation. Black dashed lin! es, the curve of RAUC = T; red dashed lines, the curve of RAUC = T2. () The simulated relationship between gene expression output and oscillatory input pulse number (n). Black dashed lines, the curve of RAUC = n; red dashed lines, the curve of RAUC = n2. Left: we set k2 = ω · k1 and changed k2 + ω · k1 to the equations used in simulations as indicated. With increasing frequency (pulse number), the interval duration changes from smaller than 1/Ton to larger than 1/Ton. Right: we set k2 + ω · k1 = 0.1 × (1/Ton) and changed Toff to the equations used in simulations as indicated. The variables k2, ω, k1 and Ton are fixed. In this case, pulse number does not correlate with pulse frequency. * Figure 7: Microarray analysis to evaluate the model predictions. () Msn2 nuclear localization response to different 1-NM-PP1 treatments: red line, 120 nM 1-NM-PP1, 20 min; green line, 3 μM 1-NM-PP1, 20 min; blue line, 3 μM 1-NM-PP1, 40 min; orange line, 750 nM 1-NM-PP1, 5 min × 3; black line, 750 nM 1-NM-PP1, 5 min × 6. () Measured time courses of mRNA levels from representative target genes (solid circles: normalized fold change of mRNA level with baseline subtracted). The inputs in panel were used experimentally to produce the measured mRNA time traces (the input and the corresponding response use the same color). () Distributions of Msn2 binding sites (red) relative to the experimentally determined nucleosome profile (blue, data not shown) within promoters of target genes. The averaged nucleosome profiles were obtained by dividing the sum of nucleosome positioning signals of all genes in one group by the gene numbers. The distribution of Msn2 binding sites (5′-AGGGG-3′ or 5′-CCCCT-3′) is represented by bars corresponding to! the sum of the numbers of Msn2 binding sites in each ten-base-pair window. () mRNA ratios of target genes in different encoding regimes (blue, Group I genes; red, Group II genes). The mRNA ratio of each gene is calculated by dividing the area under the curve of the mRNA time course (which correlates with gene expression level, Supplementary Fig. 7) at low transcription factor inputs by the area under the curve at high inputs. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Gene Expression Omnibus * GSE32703 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA. * Nan Hao & * Erin K O'Shea * Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA. * Nan Hao & * Erin K O'Shea * Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. * Nan Hao & * Erin K O'Shea * Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. * Erin K O'Shea Contributions N.H. and E.K.O. designed the project. N.H. carried out the experiments and analyzed the data. N.H. and E.K.O. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Erin K O'Shea Author Details * Nan Hao Search for this author in: * NPG journals * PubMed * Google Scholar * Erin K O'Shea Contact Erin K O'Shea Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (7M) Supplementary Figures 1–15, Supplementary Results and Supplementary Methods Audio files * Supplementary Video 1 (3M) Time-lapse video of Msn2-YFP in response to 0.1% glucose limitation. * Supplementary Video 2 (3M) Time-lapse video of Msn2-YFP in response to 0.375 M KCl. * Supplementary Video 3 (4M) Time-lapse video of Msn2-YFP in response to 0.01 mM H2O2. * Supplementary Video 4 (2M) Time-lapse video of Msn2-YFP in response to oscillatory 1-NM-PP1 treatment. Additional data Entities in this article * Zinc finger protein MSN2 MSN2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Transcriptional regulator CRZ1 CRZ1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Zinc finger protein MSN4 MSN4 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * cAMP-dependent protein kinase type 1 TPK1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * cAMP-dependent protein kinase type 2 TPK2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * cAMP-dependent protein kinase type 3 TPK3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Glutaredoxin-1 GRX1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Uncharacterized membrane protein YLR312C YLR312C Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Cellular tumor antigen p53 TP53 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia - Ndc10 is a platform for inner kinetochore assembly in budding yeast
- Nat Struct Mol Biol 19(1):48-55 (2012)
Nature Structural & Molecular Biology | Article Intrinsic tethering activity of endosomal Rab proteins * Sheng-Ying Lo1, 2 * Christopher L Brett1, 5 * Rachael L Plemel1 * Marissa Vignali3 * Stanley Fields3, 4 * Tamir Gonen1, 4, 5 * Alexey J Merz1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:40–47Year published:(2012)DOI:doi:10.1038/nsmb.2162Received 30 May 2010 Accepted 22 September 2011 Published online 11 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Rab small G proteins control membrane trafficking events required for many processes including secretion, lipid metabolism, antigen presentation and growth factor signaling. Rabs recruit effectors that mediate diverse functions including vesicle tethering and fusion. However, many mechanistic questions about Rab-regulated vesicle tethering are unresolved. Using chemically defined reaction systems, we discovered that Vps21, a Saccharomyces cerevisiae ortholog of mammalian endosomal Rab5, functions in trans with itself and with at least two other endosomal Rabs to directly mediate GTP-dependent tethering. Vps21-mediated tethering was stringently and reversibly regulated by an upstream activator, Vps9, and an inhibitor, Gyp1, which were sufficient to drive dynamic cycles of tethering and detethering. These experiments reveal a previously undescribed mode of tethering by endocytic Rabs. In our working model, the intrinsic tethering capacity Vps21 operates in concert with convent! ional effectors and SNAREs to drive efficient docking and fusion. View full text Figures at a glance * Figure 1: GTP-bound Vps21 tethers liposomes. () Experimental configuration. Full details are in Online Methods and Supplementary Methods. () Liposome particle size distributions were measured by QLS after 60-min incubation in the presence of the indicated Rab-His10 proteins, preloaded with GDP or GTP. Error bars indicate mean and s.e.m. for three independent experiments. () TEM images of negatively stained samples taken from experiment in . () Liposomes were prepared as in –, except that fluorescent lipid was incorporated. Liposomes were incubated for 20 or 40 min, then a drop of the suspension was imaged by epifluorescence microscopy (200 ms exposure). Brightness and contrast adjustments are identical for the panels shown. Traces below the images show pixel intensities along the indicated dashed lines (AU, arbitrary units). Untethered liposomes are small and move rapidly and appear as diffuse fluorescence. As tethering proceeds, clusters grow in size and the fluorescent background markedly decreases indicating that ! most individual liposomes in the population have tethered. Liposomes with GDP- His10-Vps21 are shown at 20 min incubation and were indistinguishable from those at 40 min incubation. * Figure 2: Vps21 surface density and tethering activity. (,) Liposome tethering, measured by QLS, was examined as a function of Vps21 membrane surface density. Vps21 was loaded with GTP () or GDP (). Insets, onset of tethering at low Vps21 surface densities. Additional surface density data for Vps21 and Ypt7 are in Supplementary Figure 1. () To test effect of ionic strength, liposomes were decorated with Vps21-GDP or Vps21-GTP at two different surface densities, and tethering was monitored by QLS in buffers containing indicated salt concentrations. As in ,, indicated Vps21 surface densities are upper-bound estimates. Data are mean and s.e.m. from three independent experiments. * Figure 3: Vps21 interactions in trans are required for efficient tethering. () Schematic of two possible mechanisms of Rab-mediated tethering. () Schematic of bead-liposome tethering assay. () GTP-loaded GST–Vps21 beads were photographed after incubation for 20 or 60 min in presence of fluorescent liposomes containing 6 mol% Ni2+-NTA-DOGS and GTP-loaded Vps21-His10. Images are representative of nine independent experiments. () As in (inset) but without GTP-loaded Vps21-His10. () As in (inset), but liposomes were prepared without Ni2+-NTA-DOGS. () As in , except that after 20 min incubation, 10 mM reduced glutathione was added (left), or buffer without glutathione was added (right). Samples were then incubated for 4 min more, then photographed. Scale bars, 15 μm () and 75 μm (–). * Figure 4: The Vps21 C-terminal linker is not required for tethering. () Vps21-His10 fusion proteins lacking last 10, 20 or 30 residues of the Vps21 C-terminal linker were prepared. Purified proteins (5 μg) were analyzed by SDS-PAGE. () Liposomes bearing these proteins were assayed by QLS for the ability to drive tethering over indicated range of surface densities. Each construct was loaded with either GTP (filled symbols) or GDP (open symbols). Liposomes contained 4.5 mol% Ni2+-NTA-DOGS. Error bars indicate mean and s.e.m. from four independent experiments. * Figure 5: Vps21-GTP interacts with known effectors and with itself in yeast two-hybrid assays. A positive interaction in the yeast two-hybrid assay is indicated by yeast colony growth on medium lacking tryptophan, leucine and histidine, and supplemented with 1.5 mM 3-aminotriazole. The Vps21 effectors Vac1 (also known as Pep7), Vps3 and Vps8 are positive controls for interaction selectivity with Vps21-GTP, whereas Vps9 is a control for interaction selectivity with Vps21-GDP. * Figure 6: Vps21 interacts with Ypt53 and Ypt10 to drive GTP-dependent heterotypic tethering. () Heterotypic Rab-Rab tethering was assayed as in Figure 3 except that beads were decorated with various GTP-loaded GST-Rab fusion proteins, as indicated. Bottom, representative fields of beads under epifluorescence illumination. Top, fluorescence intensity profile plots of representative beads. () Assays were done as in , except that the Rabs were preloaded with either GTP or GDP. Ypt6, which does not interact with Vps21, was a negative control. Scale bars, 75 μm. * Figure 7: Regulation and reversibility of Vps21-mediated liposome tethering. () GEF-stimulated tethering. Tethering by GDP-loaded Vps21-decorated liposomes was measured by QLS after addition of 0.2 mM GTP and varying concentrations of Vps9. Data are mean and s.e.m.; data points from three independent experiments were binned into 10-min intervals. () GAP-mediated reversal of tethering. GTP-loaded Vps21-decorated liposome tethering was measured by QLS after addition of Gyp1TBC or Gyp1TBC-R343K. Error bars indicate mean and s.e.m.; data from three independent experiments were binned into 2-min intervals. () Regulated cycle of tethering and detethering. Vps21-mediated liposome tethering, measured by QLS, was examined during sequential addition of 20 μM GTP, 5 μM Vps9 and 10 μM Gyp1TBC. Data are representative of three independent experiments. () Histograms of Vps21-decorated liposome size distributions, derived from QLS, at time points indicated in . () TEM images of negatively stained samples withdrawn at indicated time points from experiment analyze! d in ,. * Figure 8: Model for Rab-Rab driven tethering in endosome docking and fusion. In this working model, three representative Rab functions are shown: classical effector-mediated tethering, Rab-Rab tethering and coordination of trans-SNARE complex assembly by a Rab-mediated recruitment of a SNARE-binding regulator. Together, these mechanisms could in principle coordinate an ordered tethering, docking and fusion sequence. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biochemistry, University of Washington School of Medicine, Seattle, Washington, USA. * Sheng-Ying Lo, * Christopher L Brett, * Rachael L Plemel, * Tamir Gonen & * Alexey J Merz * Department of Chemistry, University of Washington, Seattle, Washington, USA. * Sheng-Ying Lo * Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington, USA. * Marissa Vignali & * Stanley Fields * Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington, USA. * Stanley Fields & * Tamir Gonen * Present addresses: Department of Biology, Concordia University, Montreal, Quebec, Canada (C.L.B.); and Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA (T.G.). * Christopher L Brett & * Tamir Gonen Contributions S.Y.L. and A.J.M. conceived the project. S.Y.L. developed and validated the QLS-based tethering system; expressed, purified and characterized proteins; prepared liposomes and carried out and interpreted all QLS tethering experiments. C.L.B. and A.J.M. conceived and C.L.B. and S.Y.L. implemented the fluorescence microscopy-based tethering assays. T.G. did the E M. S.F. and M.V. developed the high-throughput yeast two-hybrid technology, and R.L.P. and M.V. executed and interpreted yeast two-hybrid screens and assays. S.Y.L. and A.J.M. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alexey J Merz Author Details * Sheng-Ying Lo Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher L Brett Search for this author in: * NPG journals * PubMed * Google Scholar * Rachael L Plemel Search for this author in: * NPG journals * PubMed * Google Scholar * Marissa Vignali Search for this author in: * NPG journals * PubMed * Google Scholar * Stanley Fields Search for this author in: * NPG journals * PubMed * Google Scholar * Tamir Gonen Search for this author in: * NPG journals * PubMed * Google Scholar * Alexey J Merz Contact Alexey J Merz Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–4, Supplementary Tables 1–4 and Supplementary Methods Additional data Entities in this article * Vacuolar protein sorting-associated protein 21 VPS21 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ras-related protein Rab-5A RAB5A Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Vacuolar protein sorting-associated protein 9 VPS9 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * GTPase-activating protein GYP1 GYP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Early endosome antigen 1 EEA1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Thyroid receptor-interacting protein 11 TRIP11 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * ADP-ribosylation factor 1 ARF1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * GTP-binding protein YPT7 YPT7 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ras-related protein Rab-3A RAB3A Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Rab GDP-dissociation inhibitor GDI1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Rab proteins geranylgeranyltransferase component A MRS6 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Protein transport protein YIF1 YIF1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * GTP-binding protein YPT52 YPT52 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * GTP-binding protein YPT53 YPT53 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * GTP-binding protein YPT10 YPT10 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ras-related protein Rab-6A RAB6A Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * GTP-binding protein YPT6 YPT6 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ras-related protein Rab-7a RAB7A Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Vacuolar protein sorting-associated protein 3 VPS3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Vacuolar protein sorting-associated protein 8 VPS8 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Vacuolar segregation protein PEP7 PEP7 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Small COPII coat GTPase SAR1 SAR1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ras-related protein Rab-5B RAB5B Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Ras-related protein Rab-5C RAB5C Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Ras-related protein Rab-9A RAB9A Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Ras-related protein Rab-11A RAB11A Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Carboxypeptidase Y PRC1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Vacuolar protein sorting-associated protein 45 VPS45 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Rabenosyn-5 ZFYVE20 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia - X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription
- Nat Struct Mol Biol 19(1):56-61 (2012)
Nature Structural & Molecular Biology | Article Ndc10 is a platform for inner kinetochore assembly in budding yeast * Uhn-Soo Cho1 * Stephen C Harrison1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:48–55Year published:(2012)DOI:doi:10.1038/nsmb.2178Received 14 June 2010 Accepted 20 September 2011 Published online 04 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Kinetochores link centromeric DNA to spindle microtubules and ensure faithful chromosome segregation during mitosis. In point-centromere yeasts, the CBF3 complex Skp1–Ctf13–(Cep3)2–(Ndc10)2 recognizes a conserved centromeric DNA element through contacts made by Cep3 and Ndc10. We describe here the five-domain organization of Kluyveromyces lactis Ndc10 and the structure at 2.8 Å resolution of domains I–II (residues 1–402) bound to DNA. The structure resembles tyrosine DNA recombinases, although it lacks both endonuclease and ligase activities. Structural and biochemical data demonstrate that each subunit of the Ndc10 dimer binds a separate fragment of DNA, suggesting that Ndc10 stabilizes a DNA loop at the centromere. We describe in vitro association experiments showing that specific domains of Ndc10 interact with each of the known inner-kinetochore proteins or protein complexes in budding yeast. We propose that Ndc10 provides a central platform for inner-kinetocho! re assembly. View full text Figures at a glance * Figure 1: Domains of K. lactis Ndc10 and crystal structure of DI–II. () Domain organization of Ndc10; numbers show residues at the domain boundaries and are derived either from limited proteolysis or from the crystal structure. () Structure of K. lactis Ndc10 (DI–II; 1–402) with 30-bp poly(dA-dT) DNA. Domain I (N domain, residues 1–100) is in cyan, and domain II (DNA-binding domain, residues 101–402) is in dark blue. Dashed lines represent disordered residues 36–39 and 283–292. A second, symmetry-related, 15-bp DNA fragment is shown in gray. The DNA has been modeled as poly(dA-dT) (see text), with the sequences of 5′-TTAATTTATAAAATT-3′ (1–15) and 5′-AAATTTTATAAATTA-3′ (1′–15′), as indicated. () Sequence conservation of Ndc10 among point-centromere yeasts. Location of insertions (red) in S. cerevisiaeNdc10 DI–II with respect to K. lactis Ndc10 DI–II, shown on a schematic representation of the primary sequence and on a ribbon representation of the structure. All molecular illustrations were made with PyMOL (Delan! o Scientific). * Figure 2: Surface charge distribution and DNA contacts of Ndc10 DI–II. () Two views of the surface charge distribution of Ndc10 DI–II; bound DNA is shown in worm representation. () Sugar-phosphate backbone interactions. Residues involved in DNA contacts are labeled and shown in stick representation. Colors as in Figure 1b. () EMSA of wild-type and mutant Ndc10 (10% (w/v) TBE acrylamide gel stained sequentially with ethidium bromide and Coomassie blue). * Figure 3: Structural alignment of K. lactis Ndc10 DI–II with Flp recombinases. () Monomer structure of Flp (PDB 1M6X) aligned with the K. lactis Ndc10 DI–II. The N domain and the DNA-binding domain of Flp recombinase are colored in orange and yellow, respectively. In Flp, the DNA structure of the Holliday junction was replaced by 30-bp CDEIII DNA for simple comparison. () Folding diagrams of K. lactis Ndc10 DI–II and Flp recombinase. Secondary-structure elements are labeled according to their position in the polypeptide chain; domains are colored as in panel . * Figure 4: Dimerization of K. lactis Ndc10 DI–III. () Views of the probable Ndc10 DI–II dimer (symmetry axis along b in the C2221 space group). The subunits of the dimer contact different pseudocontinuous DNA duplexes. Domains I and II of the Ndc10 dimer are colored in cyan and blue for the one molecule, and green and orange for the other. () EMSA of Ndc10 DI–III with increasing amounts of 30-bp CDEIII DNA. Color code for proteins is the same as in . () DNA-capture assay with two different labels. Either Ndc10 DI–III or Ndc10 DI–II was incubated with a mixture of equal amounts of biotinylated and unmodified CDEIII DNA, the latter including 32P-labeled product (10%). (–) Ratio of Ndc10 DI–III and CDEIII DNA, as determined by analytical size-exclusion chromatography. * Figure 5: Interactions of Ndc10-associated proteins or protein complexes in the inner kinetochore. (–) Ni2+ affinity pulldown of 35S-labeled, in vitro translated prey proteins with purified, His-tagged bait protein, analyzed by SDS-PAGE and visualized by phosphoimaging. Each panel includes a lane loaded with 10% of the in vitro translation reaction mixture (to monitor extent of synthesis) and either in vitro translated maltose-binding protein as a prey or purified His-tagged MBP as a bait (negative controls). * Figure 6: Interaction of Ndc10 domain IV–V with N-terminal Scm3. () Ni2+ affinity pulldown of 35S-labeled, in vitro translated Ndc10 proteins with purified, His-tagged Scm3 proteins, analyzed by SDS-PAGE and visualized by phosphoimaging. () In vitro amylose pulldown of purified MBP-tagged Scm3 proteins with Ndc10 domain IV–V. () Schematic overview of domain association of K. lactis Ndc10 with other kinetochore proteins. Ndc10 DI interacts with CBF3 core; Ndc10 DI–II, with Cbf1 (229–359) and Bir1p (1–328). Scm3 N (1–28) associates with Ndc10 DIV–V but not with DV. Interaction of Cbf1 with Ndc10 was confirmed by analytical size-exclusion chromatography using purified proteins (Supplementary Fig. 6). * Figure 7: Schematic model of Ndc10 interactions on budding yeast centromeres. Cbf1 and CBF3 core recognize CDEI and CDEIII, respectively. Ndc10 does not have sequence-specific DNA contacts, but it binds in defined register through its interactions with Cbf1 and CBF3 core. We propose that these contacts bring CDEI and CDEIII together to form a loop. Two potential loop configurations are shown. The Scm3–Cse4–H4 heterotrimeric complex can be recruited through Scm3–Ndc10 interaction. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3SQI * 3T79 * 3SQI * 3T79 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Jack and Eileen Connors Structural Biology Laboratory and Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. * Uhn-Soo Cho & * Stephen C Harrison Contributions U.-S.C. designed and conducted experiments, determined and refined the structures, analyzed data and wrote the manuscript; S.C.H directed the project, analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Stephen C Harrison Author Details * Uhn-Soo Cho Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen C Harrison Contact Stephen C Harrison Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (10.1 MB) Supplementary Figures 1–8 and Supplementary Methods Additional data Entities in this article * Suppressor of kinetochore protein 1 SKP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Centromere DNA-binding protein complex CBF3 subunit C CTF13 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Centromere DNA-binding protein complex CBF3 subunit B CEP3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Centromere DNA-binding protein complex CBF3 subunit A CBF2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Histone H3-like centromeric protein A CENPA Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Histone H4 Homo sapiens * View in UniProt * View in Antibodypedia * Histone H3-like centromeric protein CSE4 CSE4 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Protein MIF2 MIF2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Centromere protein C 1 CENPC1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Centromere-binding protein 1 CBF1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Protein SCM3 SCM3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * S-phase kinase-associated protein 1 SKP1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * S-phase kinase-associated protein 2 SKP2 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Cullin-1 CUL1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Spindle assembly checkpoint kinase IPL1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Inner centromere protein-related protein SLI15 SLI15 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Protein BIR1 BIR1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * N-terminal-borealin-like protein NBL1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Aurora kinase B AURKB Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Inner centromere protein INCENP Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Baculoviral IAP repeat-containing protein 5 BIRC5 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Borealin CDCA8 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Histone H3-like centromeric protein CSE4 KLLA0C12529g Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) * View in UniProt * View in Entrez Gene * Site-specific recombinase Flp Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * Recombinase cre cre Enterobacteria phage P1 * View in UniProt * View in Entrez Gene * Centromere-associated factor Kluyveromyces lactis * View in UniProt * Centromere-binding protein 1 Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) * View in UniProt * Holliday junction recognition protein HJURP Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Histone H4 Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) * View in UniProt - An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains
- Nat Struct Mol Biol 19(1):62-71 (2012)
Nature Structural & Molecular Biology | Article X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription * Eda Yildirim1, 2, 3, 4 * Ruslan I Sadreyev1, 2, 3, 4 * Stefan F Pinter1, 2, 3 * Jeannie T Lee1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:56–61Year published:(2012)DOI:doi:10.1038/nsmb.2195Received 11 October 2011 Accepted 08 November 2011 Published online 04 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Dosage compensation in mammals occurs at two levels. In addition to balancing X-chromosome dosage between males and females via X inactivation, mammals also balance dosage of Xs and autosomes. It has been proposed that X-autosome equalization occurs by upregulation of Xa (active X). To investigate mechanism, we perform allele-specific ChIP-seq for chromatin epitopes and analyze RNA-seq data. The hypertranscribed Xa demonstrates enrichment of active chromatin marks relative to autosomes. We derive predictive models for relationships among Pol II occupancy, active mark densities and gene expression, and we suggest that Xa upregulation involves increased transcription initiation and elongation. Enrichment of active marks on Xa does not scale proportionally with transcription output, a disparity explained by nonlinear quantitative dependencies among active histone marks, Pol II occupancy and transcription. Notably, the trend of nonlinear upregulation also occurs on autosomes. Th! us, Xa upregulation involves combined increases of active histone marks and Pol II occupancy, without invoking X-specific dependencies between chromatin states and transcription. View full text Figures at a glance * Figure 1: Allele-specific ChIP-seq. () Profiles for Pol II-S2P, H3K4me3 and H3K36me3 are mapped to M. castaneus (cast) or M. musculus (mus) alleles for two imprinted loci, Zim1 (ref. 47) and Peg3 (ref. 48) on chromosome 7. Composite tracks (comp) represent combination of cast, mus and neutral reads. Coverage values are normalized by input and are indicated on the y axis. () X chromosome shows a strong allelic skew in the occupancy of active histone marks and Pol II at the TSS and across the gene body. Bar plots show mean composite densities of Pol II-S5P, Pol II-S2P, H3K4me3 and H3K36me3 on autosomes (A) and X chromosome (X), with proportion of allelic coverage indicated by red (cast; active X) and blue (mus; inactive X) fractions. * Figure 2: Distributions of coverage densities for Pol II and active histone modifications on X chromosome and autosomes. Coverage density values are shown for H3K4me3 and Pol II-S5P at the TSS and for H3K36me3 and Pol II-S2P across the gene bodies as indicated. Distributions are plotted for actively transcribed (HCP+LCP, HCP and LCP) genes. Black line, autosomal genes; red line, X-linked genes. * Figure 3: Relationships between levels of gene expression, Pol II and active histone modifications. M. castaneus alleles of actively expressed autosomal HCP genes are represented as points, with point density shown by colored contour. Black line contour represents active HCP X-linked M. castaneus alleles (Xa). Expression, Pol II and histone modification levels are positively correlated, the relationships are nonlinear, and X-linked genes follow autosomal trends of dependency, albeit with a shift to higher values. (,) Pol II densities at the TSS () and across the gene body () versus expression (log-log scale). () H3K4me3 densities at the TSS versus expression (log-log scale). () H3K36me3 densities across the gene body versus expression (linear-log scale). () H3K4me3 versus Pol II densities at the TSS (log-log scale). (f H3K36me3 densities across the gene body versus Pol II densities at the TSS (linear-log scale). () H3K4me3 densities at the TSS versus Pol II densities across the gene body (log-log scale). () H3K36me3 versus Pol II densities across the gene body (linear-log ! scale). Decimal logarithms are used. * Figure 4: Autosomal relationships between active histone modifications, Pol II and expression are predictive of X-linked gene expression. () Actively expressed X-linked and autosomal genes show similar patterns of correlation between the levels of active marks and expression. Pearson correlation coefficients between the levels of all marks and expression (FPKM) are shown as heat maps for actively expressed (HCP+LCP, HCP and LCP) genes. In each plot, autosomal and X chromosome correlations are shown above and below diagonal, respectively. () Active X chromosome loci (X) and the corresponding set of autosomal loci (A) show similar nonlinear relationship between active marks and expression (blue curve), which produces a large average expression change in response to smaller changes in the mark occupancy (schematic). () Scatter plot of X-linked gene expression values predicted from autosome-based full linear model versus observed X-linked expression (decimal log-log scale). Shades of blue indicate point density. Identity line y = x is shown in red. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE33823 Sequence Read Archive * SRA010053 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Eda Yildirim & * Ruslan I Sadreyev Affiliations * Howard Hughes Medical Institute, Boston, Massachusetts, USA. * Eda Yildirim, * Ruslan I Sadreyev, * Stefan F Pinter & * Jeannie T Lee * Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA. * Eda Yildirim, * Ruslan I Sadreyev, * Stefan F Pinter & * Jeannie T Lee * Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. * Eda Yildirim, * Ruslan I Sadreyev, * Stefan F Pinter & * Jeannie T Lee Contributions E.Y. and J.T.L. designed the research; E.Y. and S.F.P. conducted ChIP-seq experiments; R.I.S. performed the bioinformatics analysis; S.F.P. designed the allele-specific ChIP-seq strategy and performed allele-specific alignments; E.Y., R.I.S., S.F.P., and J.T.L. analyzed the data; and E.Y., R.I.S., and J.T.L. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jeannie T Lee Author Details * Eda Yildirim Search for this author in: * NPG journals * PubMed * Google Scholar * Ruslan I Sadreyev Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan F Pinter Search for this author in: * NPG journals * PubMed * Google Scholar * Jeannie T Lee Contact Jeannie T Lee Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (16.4 MB) Supplementary Figures 1–4 and Supplementary Tables 1–3 Additional data Entities in this article * Zinc finger, imprinted 1 Zim1 Mus musculus * View in UniProt * View in Entrez Gene * Paternally-expressed gene 3 protein Peg3 Mus musculus * View in UniProt * View in Entrez Gene * Males-absent on the first protein mof Drosophila melanogaster * View in UniProt * View in Entrez Gene * RNA on the X 1 roX1 Drosophila melanogaster * View in Entrez Gene * RNA on the X 2 roX2 Drosophila melanogaster * View in Entrez Gene * Inactive X specific transcripts Xist Mus musculus * View in Entrez Gene * Proto-oncogene c-Fos Fos Mus musculus * View in UniProt * View in Entrez Gene * Transcription factor AP-1 Jun Mus musculus * View in UniProt * View in Entrez Gene - Mechanism of mismatch recognition revealed by human MutSβ bound to unpaired DNA loops
- Nat Struct Mol Biol 19(1):72-78 (2012)
Nature Structural & Molecular Biology | Article An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains * Julien D F Licchesi1 * Juliusz Mieszczanek1 * Tycho E T Mevissen1 * Trevor J Rutherford1 * Masato Akutsu1 * Satpal Virdee1, 3 * Farid El Oualid2 * Jason W Chin1 * Huib Ovaa2 * Mariann Bienz1 * David Komander1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:62–71Year published:(2012)DOI:doi:10.1038/nsmb.2169Received 07 April 2011 Accepted 29 September 2011 Published online 11 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Eight different types of ubiquitin linkages are present in eukaryotic cells that regulate diverse biological processes. Proteins that mediate specific assembly and disassembly of atypical Lys6, Lys27, Lys29 and Lys33 linkages are mainly unknown. We here reveal how the human ovarian tumor (OTU) domain deubiquitinase (DUB) TRABID specifically hydrolyzes both Lys29- and Lys33-linked diubiquitin. A crystal structure of the extended catalytic domain reveals an unpredicted ankyrin repeat domain that precedes an A20-like catalytic core. NMR analysis identifies the ankyrin domain as a new ubiquitin-binding fold, which we have termed AnkUBD, and DUB assays in vitro and in vivo show that this domain is crucial for TRABID efficiency and linkage specificity. Our data are consistent with AnkUBD functioning as an enzymatic S1′ ubiquitin-binding site, which orients a ubiquitin chain so that Lys29 and Lys33 linkages are cleaved preferentially. View full text Figures at a glance * Figure 1: Structure and specificity of an extended TRABID OTU domain. () Schematic representation of the functional domains of TRABID (top) and species conservation derived from a multiple sequence alignment (http://www.ensembl.org) (middle). An extended catalytic OTU domain was analyzed (residues 245–697, bottom). () Linkage specificity of the extended catalytic OTU domain of TRABID using diubiquitin molecules of all eight linkage types, analyzed as reported before26. TRABID was incubated with diubiquitin for the indicated times, and the reaction mixtures were resolved on an SDS-PAGE gel and silver stained. Ub, ubiquitin. () Structure of the extended TRABID OTU domain. The catalytic core is colored in shades of blue, where dark blue indicates the minimal OTU core domain, and the lighter blue indicates additional secondary structure elements found in the A20-like subfamily of OTU DUBs. The ankyrin repeats are shown in two shades of orange. The catalytic triad residues are indicated in ball-and-stick representation. () Structure of A20 (green! , left, PDB 2VFJ23) and superposition with TRABID (blue, right). () Catalytic triad residues of TRABID are shown in ball-and-stick representation with yellow sulfur, red oxygen and blue nitrogen atoms. A red sphere indicates a water molecule, and yellow dotted lines indicate hydrogen bonds. A 2Fo – Fc electron density map contoured at 1σ covers relevant residues. () The A20 catalytic triad is shown, with atoms colored as in . * Figure 2: TRABID contains two ankyrin repeats with roles in ubiquitin binding. () Structure of the ankyrin domain in TRABID showing the two repeats. () Structure of RNase L (PDB 1WDY47), the ankyrin-repeat protein with highest similarity to the TRABID ankyrin domain in a DALI search (Z score 8.4). The eight ankyrin repeats are numbered. () Superposition of the TRABID ankyrin domain and RNase L. () The minimal OTU domain of yeast Otu1 (green) with ubiquitin (yellow) bound at the S1 site (PDB 3BY4 (ref. 25)). The orientation matches that of the minimal OTU domain core indicated in Figure 1c. Ub, ubiquitin. () Superposition of TRABID and yeast Otu1 reveals the relative position of the S1 ubiquitin-binding site on TRABID, and this suggests that the ankyrin domain may constitute an S1′ ubiquitin-binding site. * Figure 3: A conserved hydrophobic surface on AnkUBD binds ubiquitin. () 1H-15N HSQC spectrum of 13C-15N–labeled TRABID ankyrin domain. () Closeup of the region within the red box in , showing resonances of the doubly labeled ankyrin domain (blue) and their shifts upon addition of 250 μM (yellow) or 1 mM (red) unlabeled ubiquitin (Ub). Arrows indicate the shift of individual resonances. () Weighted chemical shift perturbation map of the AnkUBD binding to ubiquitin. () AnkUBD residues are colored according to the degree of perturbation from blue (unperturbed) to red (strongly perturbed), and crucial residues are shown in stick representation. () The AnkUBD surface is shown colored as in and key residues are labeled. () Invariant residues derived from a species sequence alignment (Supplementary Fig. 2b) are shown in red on a white AnkUBD surface. * Figure 4: AnkUBD binds the ubiquitin hydrophobic patch. Ubiquitin (Ub) binding to the AnkUBD was confirmed by NMR shift mapping experiments, for which spectra of 15N ubiquitin were recorded in the absence and presence of AnkUBD (Supplementary Fig. 3). () Perturbation of a selected resonance (that of ubiquitin Leu43, yellow) by titration of increasing concentrations of AnkUBD (colored from red to cyan) is shown as an example. The complete spectra can be found in Supplementary Figure 3. () The resulting weighted chemical shift perturbation map reveals a familiar pattern seen when proteins bind to the ubiquitin hydrophobic patch. () Ubiquitin residues are colored according to the degree of perturbation from blue (unperturbed) to red (strongly perturbed) and crucial residues are shown in stick representation. () The ubiquitin surface is shown colored as in and crucial residues are labeled. () Titration experiments using indicated concentrations of AnkUBD mutants H317A (left), I320D (middle) and L332E (right) were conducted. The same ! resonance as in is shown. Mutant L332E did not perturb any resonances, whereas H317A and I320D perturbed a few resonances to a lesser degree (Supplementary Fig. 3). * Figure 5: Analysis of TRABID DUB activity. () Bacterial TRABID variants were incubated with polyubiquitin substrates for indicated times and visualized by silver staining. Comparison of activity and specificity of the isolated OTU domain (above, [E] 1.2 μM) with TRABID AnkOTU (below, [E] 0.2 μM, reproduced from Fig. 1b to allow comparison). Input enzyme levels are shown in OTU panel (see also Supplementary Fig. 4a). Ub, ubiquitin. (–) Mammalian TRABID variants were incubated with polyubiquitin substrates for indicated times and visualized by silver staining. Flag-tagged TRABID variants were purified from HEK293 cells and used in DUB assays. () Specificity of mammalian full-length (FL), AnkOTU and OTU TRABID against the diubiquitin panel after overnight (O/N, 16 h) incubation. () Time-course analysis of mammalian TRABID variants against its substrate linkages. FL ΔAnk means full-length, lacking AnkUBD. Input (Inp) controls highlight the stability of ubiquitin substrates in the absence of enzyme in the reaction mi! xture. () Activity of full-length TRABID with point mutations in the AnkUBD against its preferred diubiquitin substrates. Full-length C443S, catalytic mutant. DUB activity assays carried out with material obtained from Flag-empty vector (ev) immunoprecipitation showed no activity. () Time-course activity of TRABID variants against Lys63-linked hexaubiquitin. * Figure 6: Role of the NZF domains in cleaving longer ubiquitin chains. Mammalian TRABID variants were incubated with polyubiquitin substrates for indicated times and visualized by silver staining. () Activity of TRABID variants against Lys29, Lys33 and Lys63-linked diubiquitin (Ub2) at indicated time point. Full-length C443S, catalytic mutant; full-length NZFmut, full-length with mutations in all three NZF domains; FL ΔAnk, full-length, lacking AnkUBD; AnkOTU, crystallized fragment; OTU, OTU domain. () Time-course analysis of mammalian full-length TRABID, full-length NZFmut and AnkOTU activity toward Lys63-linked hexaubiquitin. () Model for the role of the AnkUBD as an S1′ ubiquitin-binding site in TRABID. () Model for the additional contribution of the NZF domains in cleaving longer polyubiquitin chains. * Figure 7: In vivo DUB assay NZF and AnkUBD are essential for TRABID puncta. () Localization studies with GFP-TRABID in COS-7 cells 18 h after transfection (left). Nuclei are stained using DAPI (middle). The right image is a merge of the channels. The domain structure of TRABID is shown above. () A GFP-tagged full-length TRABID catalytic mutant (C443S; a yellow star in the domain representation indicates the mutation) adopts a punctate localization in COS-7 (shown) and other cell types35. () FRAP experiments on a control (black) or puncta-containing volume (C443S, blue). Fluorescent recovery is recorded over time. () Localization studies of TRABID GFP-tagged AnkOTU C443S and GFP-tagged full-length NZFmut C443S, colored as in . The domain structure is shown (left), and the GFP fluorescence of either construct (right) shows that no puncta are formed. Ub, ubiquitin. () Puncta-forming GFP-tagged C443S (left image) was coexpressed with Flag-ubiquitin or single-lysine ('Konly') ubiquitin mutants (middle image). The merged image is shown to the right. Furth! er ubiquitin mutants are shown in Supplementary Figure 6a. (–) Dissolving TRABID assemblies requires the AnkUBD. () Puncta-forming GFP-tagged full-length TRABID C443S (left) was expressed in COS-7 cells and TRABID assemblies were visualized (left). Nuclei are stained using DAPI (middle). The merged image is shown to the right. (–) As in , but in addition, Flag-tagged full-length WT TRABID constructs (), full-length NZFmut () or full-length ΔAnk () were coexpressed (far left), and the presence of GFP puncta was assessed. Additional data are shown in Supplementary Figure 6b. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3ZRH * 3ZRH Referenced accessions Protein Data Bank * 2VFJ * 1WDY * 3BY4 * 2VFJ * 1WDY * 3BY4 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. * Julien D F Licchesi, * Juliusz Mieszczanek, * Tycho E T Mevissen, * Trevor J Rutherford, * Masato Akutsu, * Satpal Virdee, * Jason W Chin, * Mariann Bienz & * David Komander * Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands. * Farid El Oualid & * Huib Ovaa * Present address: Scottish Institute for Cell Signalling, Protein Ubiquitylation Unit, University of Dundee, Dundee, UK. * Satpal Virdee Contributions D.K., M.B. and J.D.F.L. designed the research. J.D.F.L., D.K., J.M., T.E.T.M., T.J.R. and M.A. conducted the experiments. F.E., H.O., S.V. and J.W.C. contributed reagents. D.K. wrote the manuscript, with help from all authors. Competing financial interests H.O. and F.E. are cofounders of UbiQ Bio BV. Corresponding author Correspondence to: * David Komander Author Details * Julien D F Licchesi Search for this author in: * NPG journals * PubMed * Google Scholar * Juliusz Mieszczanek Search for this author in: * NPG journals * PubMed * Google Scholar * Tycho E T Mevissen Search for this author in: * NPG journals * PubMed * Google Scholar * Trevor J Rutherford Search for this author in: * NPG journals * PubMed * Google Scholar * Masato Akutsu Search for this author in: * NPG journals * PubMed * Google Scholar * Satpal Virdee Search for this author in: * NPG journals * PubMed * Google Scholar * Farid El Oualid Search for this author in: * NPG journals * PubMed * Google Scholar * Jason W Chin Search for this author in: * NPG journals * PubMed * Google Scholar * Huib Ovaa Search for this author in: * NPG journals * PubMed * Google Scholar * Mariann Bienz Search for this author in: * NPG journals * PubMed * Google Scholar * David Komander Contact David Komander Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–6 and Supplementary Methods Additional data Entities in this article * Ubiquitin thioesterase ZRANB1 ZRANB1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Catenin beta-1 CTNNB1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Ubiquitin thioesterase OTUB1 OTUB1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Ubiquitin thioesterase OTUB2 OTUB2 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Tumor necrosis factor alpha-induced protein 3 TNFAIP3 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Ubiquitin thioesterase OTU1 OTU1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Transitional endoplasmic reticulum ATPase VCP Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * OTU domain-containing protein 7B OTUD7B Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * AMSH-like protease STAMBPL1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * OTU domain-containing protein 5 OTUD5 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Ubiquitin carboxyl-terminal hydrolase 5 USP5 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * E3 ubiquitin-protein ligase UBR5 UBR5 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * 2-5A-dependent ribonuclease RNASEL Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia - The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β1−40 peptide
- Nat Struct Mol Biol 19(1):79-83 (2012)
Nature Structural & Molecular Biology | Article Mechanism of mismatch recognition revealed by human MutSβ bound to unpaired DNA loops * Shikha Gupta1 * Martin Gellert1 * Wei Yang1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:72–78Year published:(2012)DOI:doi:10.1038/nsmb.2175Received 09 August 2011 Accepted 12 October 2011 Published online 18 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg DNA mismatch repair corrects replication errors, thus reducing mutation rates and microsatellite instability. Genetic defects in this pathway cause Lynch syndrome and various cancers in humans. Binding of a mispaired or unpaired base by bacterial MutS and eukaryotic MutSα is well characterized. We report here crystal structures of human MutSβ in complex with DNA containing insertion-deletion loops (IDL) of two, three, four or six unpaired nucleotides. In contrast to eukaryotic MutSα and bacterial MutS, which bind the base of a mismatched nucleotide, MutSβ binds three phosphates in an IDL. DNA is severely bent at the IDL; unpaired bases are flipped out into the major groove and partially exposed to solvent. A normal downstream base pair can become unpaired; a single unpaired base can thereby be converted to an IDL of two nucleotides and recognized by MutSβ. The C-terminal dimerization domains form an integral part of the MutS structure and coordinate asymmetrical ATP hyd! rolysis by Msh2 and Msh3 with mismatch binding to signal for repair. View full text Figures at a glance * Figure 1: Overall structures of MutSβ–DNA complexes. () Orthogonal views of Loop3 structure in ribbon diagrams, with MSH2 in green and MSH3 in blue. The DNA is shown in a space-filling model with the backbone in red, bases in light pink, and the unpaired nucleotides in yellow and orange. ADP bound to MSH2 is shown in purple sticks. () Side views of DNA-binding domains and DNA in Loop2, Loop4 and Loop6 structures. Each unpaired CA dinucleotide repeat is shown in yellow and orange. Domain I of MSH3 is the MBD. MSH2 and MSH3 subunits are indicated by circled numbers 2 and 3, respectively. Domains I–V and dimerization domains are indicated. * Figure 2: Comparison of MutSα and MutSβ proteins. () Ribbon diagram of MSH2 from MutSβ. Domains I, II, III, IV, V and the DMDs are shown in blue, green, yellow, orange, pink and red, respectively. MSH2 of MutSα is superimposed and shown in gray. Domain interfaces between I and II and between II, III and V are highlighted in magenta. () Ribbon diagram of MSH3 in the same orientation and same color codes. Domains I (MBD) and IV (clamp) interact as indicated by the dotted oval. () Superposition of the mismatch-binding subunits in MutSα, MutSβ, E. coli and TaqMutS in the same orientation as in and . Except for domain IV, they superimpose very well. () A ribbon diagram of the interface between domains I in MutSβ. Protein residues in all figures are labeled in one-letter code for clarity. () On the left is a ribbon diagram of MSH3 MBD decorated by residues conserved among MutS homologs (shown as yellow-blue-red stick-and-ball models) and residues unique among MSH3 homologs (pink-blue-red stick-and-ball models). On the right ! is the superposition of MSH3 (blue) and MSH6 (gray) MBDs. The r.m.s. deviation between them is 0.7 Å over 87 pairs of Cα atoms. * Figure 3: IDL recognition by MutSβ. () A closeup comparison of MSH3–IDL interaction and TaqMutS with a single unpaired base (ΔT). () DNA-binding domains and DNA in Loop4. Domains I and IV of MSH2 are shown in green and yellow, and MBD and clamp domains of MSH3 in blue and orange, respectively. () Diagram of the protein-DNA interactions using the same color scheme as in . () Space-filling model of four IDLs and their interaction with domain I of MSH2 (green) and MBD of MSH3 (blue). The base pairs surrounding the IDL are shown in light (upstream) and dark (downstream) pink. For Loop2 and Loop4, a back view looking into the minor groove is also shown. * Figure 4: Dimerization domains (DMD) of MutSβ. (,) Orthogonal views of the C-terminal halves of DMDs. The hydrophobic side chains at the interface, and polar residues forming salt bridges that stabilize intrasubunit interactions, are shown as sticks with carbon in light gray, nitrogen in blue and oxygen in red. Glu901 and Lys912 of MSH2 form N- and C-caps of the MSH3 helices. () The ATPase domain (light green) and DMD (green) of MSH2 are shown with the trans-acting N2 (red and cyan) and DMD (blue) of MSH3. The ADP bound to MSH2 is shown as purple sticks. The two shaded ovals indicate the enlarged areas shown in , and . () A closeup view of the interactions between MSH3 DMD(N) and the ATPase domain of MSH2. Interactions between hydrophobic residues dominate, and two pairs of salt bridges (red dashes) may have alternative interacting partners (black dashes) if the two subunits slide relative to each other. * Figure 5: Asymmetric ATPase sites of MutSβ. () Ribbon diagram of the ATPase and dimerization domains. MSH2 is shown in light and dark green, and MSH3 in light and dark blue. The trans-acting N2 regions of MSH2 and MSH3 are highlighted in red. The aromatic side chains connecting the ATPase site to the DMD are shown as blue (MSH3) and green sticks (MSH2). () A view 180° from showing the asymmetric DMDs, biased toward the MSH2 ATPase site. () Comparison of the connection between the ATP binding site and DMD in MSH3 (blue), MSH6 with ADP (pink) and without (yellow) after superposition. The critical aromatic side chains are shown as sticks. * Figure 6: Mechanism of mismatch recognition. Msh2 is drawn in green and Msh3 and Msh6 in blue. The ATPase activities of the two subunits are indicated by the curved arrows: the thicker the arrow, the higher the activity. The DNA-binding domains are flexible in the apo form. Binding to normal DNA, which is resistant to bending, induces conformational changes in the DNA-binding domains and ATP hydrolysis. But the protein-DNA association is not stable, and MutS(α or β) can dissociate from or slide along DNA. Binding to a mismatched DNA, which is readily bent, leads to stable association of Msh3 or Msh6 with DNA and inhibition of its ATPase activity. ATP binding by Msh3 or Msh6, however, leads to release of the mismatched DNA. When Msh2 is bound to ATP and Msh3 or Msh6 to the mismatched DNA, MutS(α or β) can recruit MutLα to form a 'repairosome', thus initiating the repair process. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3THY * 3THX * 3THW * 3THZ * 3THY * 3THX * 3THW * 3THZ Referenced accessions Protein Data Bank * 1EWQ * 2O8E * 2O8B * 1EWQ * 2O8E * 2O8B Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, USA. * Shikha Gupta, * Martin Gellert & * Wei Yang Contributions S.G. conducted all experiments and collected X-ray data. W.Y. determined and refined the structures. S.G., M.G. and W.Y. contributed to the experimental design, data interpretation and manuscript preparation. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Wei Yang Author Details * Shikha Gupta Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Gellert Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Yang Contact Wei Yang Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (13M) Supplementary Figures 1–7 Additional data Entities in this article * DNA mismatch repair protein mutS mutS Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA mismatch repair protein Msh2 MSH2 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * DNA mismatch repair protein Msh3 MSH3 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * DNA mismatch repair protein mutL mutL Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA mismatch repair protein Msh6 MSH6 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * DNA mismatch repair protein Msh3 Msh3 Mus musculus * View in UniProt * View in Entrez Gene * DNA mismatch repair protein Msh6 Msh6 Mus musculus * View in UniProt * View in Entrez Gene * DNA mismatch repair protein MSH3 MSH3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA mismatch repair protein MSH6 MSH6 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA mismatch repair protein mutS Thermus aquaticus * View in UniProt * DNA mismatch repair protein MSH2 MSH2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28
- Nat Struct Mol Biol 19(1):84-89 (2012)
Nature Structural & Molecular Biology | Article The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β1−40 peptide * Priyanka Narayan1 * Angel Orte1, 2 * Richard W Clarke1 * Benedetta Bolognesi1 * Sharon Hook1 * Kristina A Ganzinger1 * Sarah Meehan1 * Mark R Wilson3 * Christopher M Dobson1 * David Klenerman1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:79–83Year published:(2012)DOI:doi:10.1038/nsmb.2191Received 27 May 2011 Accepted 14 October 2011 Published online 18 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a newly-discovered risk factor in Alzheimer's disease. We have examined the interactions between human clusterin and the Alzheimer's disease–associated amyloid-β1−40 peptide (Aβ1−40), which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single-molecule fluorescence methods, we have found that Aβ1−40 forms a heterogeneous distribution of small oligomers (from dimers to 50-mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence both the aggregation and disaggregation of Aβ1−40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and Al! zheimer's disease. View full text Figures at a glance * Figure 1: Bulk and single-molecule studies of Aβ1−40. () Appearance and disappearance of species populated during the aggregation of Aβ1−40 (2 μM at 37 °C with agitation). Fibril formation monitored by thioflavin (ThT) fluorescence (top). The inset is a transmission electron microscopy (TEM) image of the fibrils present after 24 h of incubation (scale bar, 200 nm). The concentration of soluble oligomers (dimers to 50-mers; middle) and of monomeric species (bottom) are both tracked using cTCCD. The data are averaged from multiple experimental repetitions (2 μM Aβ1−40, n = 3; error bars are s.e.m.). () A representative distribution of apparent sizes of oligomers formed during Aβ1−40 aggregation and disaggregation (error bars are s.d.). Insets are zoomed into regions of dimers to 15-mers and 16-mers to 50-mers to show greater detail. () A comparison of the distributions of apparent oligomer sizes during aggregation and disaggregation experiments (2 μM Aβ1−40 aggregation, n = 3; disaggregation, n = 12; 10−30 nM A�! �1−40 aggregation, n = 4; error bars are s.e.m.). () Time dependence of the concentration of soluble species released from a pellet of fibrils (n = 12; error bars are s.e.m.). * Figure 2: The effects of clusterin on the aggregation of Aβ1−40. () Fraction of oligomers detected in solution during the aggregation of Aβ1−40 with and without clusterin (Aβ1−40 and clusterin are both at a concentration of 600 nM; n = 3 and error bars are s.e.m.). () TIRFM image of the species present after 24 h of aggregation of a 2-μM solution of Aβ1−40 without clusterin (left). TIRFM image of a 2 μM solution of Aβ1−40 after 24 h of aggregation, but with clusterin added at a concentration of 2 μM 4 h after the start of the reaction, during the fibril growth phase (right). An approximately 50% reduction in the average dimensions of species present is observed in the presence of clusterin (from 1,400 ± 200 nm without clusterin to 780 ± 60 nm with clusterin, s.e.m., P-value is 0.01, two-sample independent, two-tailed t-test). Scale bars, 5 μm. () Fractions of species formed during the aggregation of a 2 μM solution that are oligomeric and that are in Aβ–clusterin complexes. (n = 3, error bars are s.e.m.). () Proporti! on of Aβ–clusterin complexes persisting at 10−20 nM (total peptide concentration) at 21 °C. Complexes were formed between clusterin and oligomers formed in both aggregation and disaggregation reactions. For both traces, n = 3 and error bars are s.e.m. There is no statistically significant change in the proportion of complexes with oligomers formed during either the disaggregation experiments (P value of 0.77, analysis of variance (ANOVA) single-factor) or the aggregation experiments (P value of 0.99, ANOVA single-factor). * Figure 3: The effects of clusterin on the disaggregation of Aβ1−40 fibrils. () Distributions of apparent sizes of oligomers formed during aggregation and disaggregation reactions with and without clusterin. (Aggregation without clusterin, n = 2 and error bars are range; aggregation with clusterin, n = 3; disaggregation without clusterin, n = 10; disaggregation with clusterin, n = 3 and error bars are s.e.m.). () Time dependence of the release of soluble species during the disaggregation experiments in the presence and absence of clusterin (top), increased oligomer concentration in the presence of clusterin in the concentration plateau region (significant, with a P value of 0.002) (bottom left) and decreased monomer concentration in the presence of clusterin, in the concentration plateau region (significant, with a P value of 0.0003) (bottom right). Both correlations were analyzed using a two-sample independent, two-tailed t-test; n = 8 and error bars are s.e.m. () TIRFM images of HiLyte488Fluor-labeled Aβ1−40 fibrils incubated overnight at 21 °C! with AlexaFluor647-labeled clusterin. Aβ1−40 fluorescence only (left), clusterin fluorescence only (middle) and colocalization of the two species (right). Scale bars, 5 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Chemistry, University of Cambridge, Cambridge, UK. * Priyanka Narayan, * Angel Orte, * Richard W Clarke, * Benedetta Bolognesi, * Sharon Hook, * Kristina A Ganzinger, * Sarah Meehan, * Christopher M Dobson & * David Klenerman * Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, Granada, Spain. * Angel Orte * School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia. * Mark R Wilson Contributions P.N., A.O., S.M., M.R.W., C.M.D. and D.K. designed the experiments. P.N. conducted the cTCCD experiments. P.N., A.O. and R.W.C. refined analysis methods. A.O. and R.W.C. developed instrumentation. R.W.C. wrote the analysis software, and designed, built and calibrated the scanning stage used for cTCCD experiments. P.N. and B.B. conducted the bulk scale experiments. P.N. and K.A.G. conducted the TIRFM experiments. P.N., B.B., K.A.G. and A.O. analyzed the data. S.H. labeled the clusterin that was purified and provided by M.R.W. All authors discussed and interpreted results and contributed to the writing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Mark R Wilson or * Christopher M Dobson or * David Klenerman Author Details * Priyanka Narayan Search for this author in: * NPG journals * PubMed * Google Scholar * Angel Orte Search for this author in: * NPG journals * PubMed * Google Scholar * Richard W Clarke Search for this author in: * NPG journals * PubMed * Google Scholar * Benedetta Bolognesi Search for this author in: * NPG journals * PubMed * Google Scholar * Sharon Hook Search for this author in: * NPG journals * PubMed * Google Scholar * Kristina A Ganzinger Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah Meehan Search for this author in: * NPG journals * PubMed * Google Scholar * Mark R Wilson Contact Mark R Wilson Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher M Dobson Contact Christopher M Dobson Search for this author in: * NPG journals * PubMed * Google Scholar * David Klenerman Contact David Klenerman Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (979K) Supplementary Figures 1–6, Supplementary Methods and Supplementary Discussion Additional data Entities in this article * Clusterin CLU Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Amyloid beta A4 protein APP Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia - Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi
- Nat Struct Mol Biol 19(1):90-97 (2012)
Nature Structural & Molecular Biology | Article Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28 * Fionna E Loughlin1 * Luca F R Gebert2 * Harry Towbin2 * Andreas Brunschweiger2 * Jonathan Hall2 * Frédéric H-T Allain1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:84–89Year published:(2012)DOI:doi:10.1038/nsmb.2202Received 20 May 2011 Accepted 10 November 2011 Published online 11 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5′-AGGAGAU-3′. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5′-NGNNG-3′ consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28. View full text Figures at a glance * Figure 1: The ZnFs of Lin28 bind single-stranded regions of pre-let-7 terminal loops. () Domain structure of Lin28 and of the construct containing the two ZnFs used in this study. () Chemically synthesized biotinylated pre-miRNAs bound by immobilized, recombinant, purified Lin28 ZnF12. Pre-let-7 family members and pre-miRNAs that are known not to be regulated by Lin28 are shown. Error bars represent s.d. of triplicate determinations. One of two assays is shown. () Upper, secondary structure of the terminal loop of pre-let-7f-1, as predicted with Mfold29. Lower left, overlay of section of 15N HSQC spectra of Lin28 ZnF12, free (gray) and bound (green) to the terminal loop of pre-let-7f-1. Arrows show the changes of selected resonances. Lower right, overlay of section of 15N HSQC spectra of Lin28 ZnF12 bound to the terminal loops of pre-let-7f-1 (green) and bound to the single-stranded 5′-AGGAGAU-3′ (purple) sequence. TL, terminal loop. * Figure 2: The solution structure of Lin28 ZnF12 bound to 5′-AGGAGAU-3′. () Representative structures of the Lin28 ZnF12–5′-AGGAGAU-3′ complex. The ZnF ribbon is shown in green, the zinc atom in purple and the RNA in yellow. () G2 recognition by ZnF2. The hydrogen bonds are indicated by dotted black lines. () G5 recognition by ZnF1. Figures were generated by MOLMOL30. Arrows identify residues; bb indicates amino acid backbone. * Figure 3: Affinity of Lin28 variants for single-stranded and pre-let-7g RNAs and processing of pre-let-7g point mutants in Huh-7 cells. () Representative ITC data obtained by titration of Lin28 ZnF12 WT and point mutant into 5′-AGGAGAU-3′ (left). Kd of Lin28 ZnF12 WT and single–amino acid mutant binding to ssRNA (right). Error bars indicate s.d. of two measurements; for details see Supplementary Table 1. () Representative inhibition curves for full-length Myc-tagged Lin28 WT and single-point mutations in HeLa cell lysates (left). Relative binding affinities (Kd) of Myc-Lin28 for pre-let-7g in HeLa cell lysates (right, average of two replicate experiments, error bars indicate s.d.). () Representative ITC data obtained upon titration of Lin28 ZnF12 into ssRNA (left). Kd of Lin28 ZnF12 binding different RNAs (right). For details see Supplementary Table 1. () Levels of mature microRNAs (let-7g, mir-16 and mir-191) in Huh-7 cells transfected with graded concentrations (10, 5 and 2.5 nM) of in vitro transcribed pre-let-7g WT or point mutants, as determined by stem-loop RT-PCR after 24 h. Mir-191 was used for! normalization and error bars indicate s.e.m. of quadruplicate determinations. * Figure 4: Comparison between the structure of Lin28 ZnFs bound to 5′-AGGAGAU-3′ (this study) and HIV nucleocapsid (NC) bound to stem-loops of the RNA packaging signal. () Lin28 bound to 5′-AGGAGAU-3′ (green) overlaid with HIV NC bound to SL3 containing a GAG loop26 (purple; PDB 1A1T). () Lin28 bound to 5′-AGGAGAU-3′ (green) overlaid with HIV NC bound to SL2 containing a GUG loop27 (blue; PDB 1F6U). () Sequence alignment of Lin28 and HIV NC, indicating the insertion of Pro158. () Representative structures of the Lin28 ZnF12–5′-AGGAGAU-3′ complex. The ZnF ribbon is shown in green, the zinc atom in purple and the RNA in yellow. () Representative structures of HIV NC–bound SL3 containing a GAG sequence in the loop. The ZnF ribbon is shown in gray, the zinc atom in purple and the RNA in yellow. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Biological Magnetic Resonance Data Bank * 17883 Protein Data Bank * 2LI8 * 2LI8 Referenced accessions Protein Data Bank * 2CQF * 1A1T * 1F6U * 2CQF * 1A1T * 1F6U Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland. * Fionna E Loughlin & * Frédéric H-T Allain * Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland. * Luca F R Gebert, * Harry Towbin, * Andreas Brunschweiger & * Jonathan Hall Contributions F.H.-T.A., F.E.L. and J.H. designed the project; F.E.L. prepared protein and RNA samples for structural studies; F.E.L. and F.H.-T.A. collected and analyzed NMR data; F.E.L. carried out the structure calculations and the ITC measurements; H.T. and A.B. did the Lin28 binding assay with miRNAs and L.F.R.G. did the quantitative PCR in cell assays. All authors discussed the results, wrote and approved the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Jonathan Hall or * Frédéric H-T Allain Author Details * Fionna E Loughlin Search for this author in: * NPG journals * PubMed * Google Scholar * Luca F R Gebert Search for this author in: * NPG journals * PubMed * Google Scholar * Harry Towbin Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Brunschweiger Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan Hall Contact Jonathan Hall Search for this author in: * NPG journals * PubMed * Google Scholar * Frédéric H-T Allain Contact Frédéric H-T Allain Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (8M) Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * Protein lin-28 homolog A LIN28A Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * GTPase KRas KRAS Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Myc proto-oncogene protein MYC Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * High mobility group protein HMGI-C HMGA2 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Ribonuclease 3 DROSHA Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Endoribonuclease Dicer DICER1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Terminal uridylyltransferase 4 ZCCHC11 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * POU domain, class 5, transcription factor 1 POU5F1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Homeobox protein NANOG NANOG Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Transcription factor SOX-2 SOX2 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Protein lin-28 homolog B LIN28B Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Protein lin-28 lin-28 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * microRNA let-7 C05G5.6 Caenorhabditis elegans * View in Entrez Gene * microRNA let-7g MIRLET7G Homo sapiens * View in Entrez Gene * microRNA let-7c MIRLET7C Homo sapiens * View in Entrez Gene * microRNA let-7f-1 MIRLET7F1 Homo sapiens * View in Entrez Gene * microRNA let-7a-1 MIRLET7A1 Homo sapiens * View in Entrez Gene * microRNA 191 MIR191 Homo sapiens * View in Entrez Gene * microRNA let-7a-3 MIRLET7A3 Homo sapiens * View in Entrez Gene * Gag-Pol polyprotein Human immunodeficiency virus type 1 group M subtype B (isolate YU-2) * View in UniProt - Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H
- Nat Struct Mol Biol 19(1):98-104 (2012)
Nature Structural & Molecular Biology | Article Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi * Caroline Thivierge1, 2, 6 * Neetha Makil1, 2, 6 * Mathieu Flamand1, 2 * Jessica J Vasale3 * Craig C Mello3, 4 * James Wohlschlegel5 * Darryl Conte Jr3 * Thomas F Duchaine1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:90–97Year published:(2012)DOI:doi:10.1038/nsmb.2186Received 25 June 2011 Accepted 14 October 2011 Published online 18 December 2011 Corrected online09 January 2012 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Endogenous RNA interference (endo-RNAi) pathways use a variety of mechanisms to generate siRNA and to mediate gene silencing. In Caenorhabditis elegans, DCR-1 is essential for competing RNAi pathways—the ERI endo-RNAi pathway and the exogenous RNAi pathway—to function. Here, we demonstrate that DCR-1 forms exclusive complexes in each pathway and further define the ERI–DCR-1 complex. We show that the tandem tudor protein ERI-5 potentiates ERI endo-RNAi by tethering an RNA-dependent RNA polymerase (RdRP) module to DCR-1. In the absence of ERI-5, the RdRP module is uncoupled from DCR-1. Notably, EKL-1, an ERI-5 paralog that specifies distinct RdRP modules in Dicer-independent endo-RNAi pathways, partially compensates for the loss of ERI-5 without interacting with DCR-1. Our results implicate tudor proteins in the recruitment of RdRP complexes to specific steps within DCR-1-dependent and DCR-1-independent endo-RNAi pathways. View full text Figures at a glance * Figure 1: Distinct DCR-1 complexes initiate endo- and exo-RNAi. () Gel filtration on wild-type embryonic extract. DRH-1, RDE-4, DCR-1, RRF-3, ERI-5, ERI-1 and DRH-3 proteins were detected by western blot on fractions from a Superose S6 column. The fractionation of molecular weight (MW) standards is indicated. The asterisk (*) labels in DRH-1 (in the low molecular weight fractions) and RDE-4 filtration panels indicate non-specific bands. () Immunoprecipitation (IP) of DCR-1, DRH-1 and RDE-4 from WT, dcr-1, rde-4 or rde-1 mutant embryos. DCR-1, RDE-1, DRH-1 and RDE-4 proteins were detected in total lysate (LOAD) and IP by western blot. Tubulin was used as a loading control. The asterisk (*) to the right of the RDE-4 panels indicates background signal from the IgG heavy chains used for immunoprecipitation, which migrate with RDE-4 around 50 kDa. () Immunoprecipitation of DRH-1 in WT and drh-1 mutant embryos. DRH-1, DCR-1, DRH-3, ERI-5 and ERI-1 were detected by western blot. The asterisks (*) to the right and left of the DRH-1 panel indicat! e non-specific bands in the loading and DRH-1 IP lanes, respectively. () Immunoprecipitation of ERI-5 in WT and eri-5 mutant embryos. DRH-3, DRH-1 and ERI-5 proteins were detected by western blot. The asterisk (*) indicates the non-specific band detected in the input lanes (LOAD) of the DRH-1 blot, as in panel . () Interaction map of the proteins detected by MuDPIT analyses in WT embryonic extracts. Proteins circled in bold (DCR-1, ERI-5, ERI-1 and RDE-4) represent immunoprecipitation targets. See Online Methods section for details on the epitope targeted. Arrowheads indicate interactions detected. The interactions of ERI-5 and ERI-1 in RDE-4 immunoprecipitation included in the diagram were only detected by western blotting. The number of interactions detected exclusively in DCR-1 or ERI-1 MuDPIT experiments is indicated (17 or 11 single target hits) and may reflect divergent functions for these proteins. * Figure 2: ERI-5 promotes the association of an RdRP module to the DCR-1 N terminus. (,) Immunoprecipitation (IP) of DCR-1 and ERI-5 in WT, eri-5, rrf-3 del (deletion mutant, pk1426), rrf-3 pm (point mutant, mg373), eri-3 and eri-1 mutant embryos. DCR-1, RRF-3, DRH-3 and ERI-5 were detected by western blotting. Tubulin was used as a loading control. () Map of the DCR-1–GST constructs used for the GST pulldown of recombinant (r) ERI-5 or ERI-3 (top). The ability of each DCR-1–GST fusion to interact with rERI-5 or rERI-3 was assessed by western blot (bottom panel) to detect recombinant rERI-5-CBP or rERI-3–Flag. The results are summarized to the right of the DCR-1 map; the minus sign denotes weak or no interaction, and the plus sign denotes an interaction (see Supplementary Fig. 2c for Coomassie blue gel staining). Percentage (%) of the loading (bottom panel) represents the fraction of rERI-5 and rERI-3 used in the GST pulldown. () ERI-3 and ERI-5 bind to DCR-1 (272–1045) simultaneously. An increasing amount of rERI-3 was incubated with DCR-1 (272–10! 45) before addition of rERI-5 and pull-down of the DCR-1 fragment. * Figure 3: ERI-5 potentiates ERI endo-RNAi small RNA biogenesis. (,) Northern () and qrtPCR analysis () of C40A11.10 26G-RNA siRNA species (siR26-1) as indicated in WT, eri-5 and rrf-3 (pk1426) mutant embryos. The C40A11.10 probe detected both 26G and 22G RNAs. 5S ribosomal RNA (rRNA) ethidium bromide staining is shown as a loading control in . The mean of at least three independent experiments is depicted as the ratio of siR26-1 or X-cluster relative to actin. Error bars indicate s.d. () Box and whisker plots show the enrichment or depletion of small RNAs targeting 26G-RNA coding genes (red) and non-annotated 26G-RNA clusters (yellow) in the indicated mutant. The left panel is an analysis of 26-nt antisense reads from embryo small RNA libraries that target the 26G-RNA loci. The right panel is an analysis of all antisense reads from adult small RNA libraries that target the 26G-RNA loci. The majority of reads in the adult samples are 22G RNAs. Values approaching 1 indicate enrichment of small RNA; values approaching 0 indicate depletion. ! Relative enrichment was calculated as the ratio of mutant per (mutant plus wild type). The top and bottom of each box represent the 75th and 25th percentiles, respectively. The horizontal line within each box represents the median value. The number of loci used to generate box and whisker plots is indicated above each plot, and the data are provided in Supplementary Data 1 and 2. * Figure 4: Tandem tudor domain proteins are required for ERI endo-siRNA biogenesis. (,) Northern () and qrtPCR () analysis of C40A11.10 26G RNAs (siR26-1) in sel-1 (RNAi) (a negative control, marked with (−)), ekl-1(RNAi), eri-5 and eri-5, and in ekl-1(RNAi) embryos. The mean of at least three independent experiments is depicted as the ratio of siR26-1 relative to actin. Error bars indicate s.d. () qrtPCR analysis of C40A11.10 26G RNAs (siR26-1) in WT, eri-5, eri-4 and double eri-5, and in eri-4 mutant embryos. The mean of at least three independent experiments is depicted as the ratio of siR26-1 relative to actin. Error bars indicate s.d. () IP of EKL-1 and DCR-1 in WT and eri-5 mutant embryos. EKL-1 and DCR-1 proteins were detected by western blot. () Immunoprecipitation of RRF-3 in WT and eri-5 mutant embryos. DCR-1, RRF-3, EKL-1 and ERI-5 proteins were detected by western blot. Tubulin was used as a loading control. * Figure 5: Roles and paralog organization of RdRP modules in ERI endo-RNAi. () IP of EKL-1 in WT and ekl-1(RNAi) (ekl-1 lanes) embryos, and immunoprecipitation of ERI-5 in WT and eri-5 mutant embryos. The RdRPs EGO-1, RRF-1 and RRF-3, and the tudor domain proteins EKL-1 and ERI-5 were detected by western blot. Asterisk (*) indicates a non-specific band. () Model of the molecular compensation of ERI-5 by EKL-1. Interactions between the RdRP module and the N-terminal helicase domain of DCR-1 couple the generation of dsRNA by RRF-3 with processive DCR-1 activity. In the eri-5 mutant, this coupling is lost and the autoinhibitory function of the helicase domain predominates, resulting in inefficient 26G-RNA production. () Paralogous RdRP modules function sequentially in ERI endo-RNAi. An RdRP module comprised of RRF-3, DRH-3 and ERI-5 together with DCR-1 function at the initial step to generate 26G RNAs, the primary siRNAs of the ERI pathway that programs ERGO-1. A paralogous RdRP module comprised of RRF-1, DRH-3 and EKL-1 is responsible for secondary si! RNA generation that is independent of DCR-1. This abundant pool of small RNAs programs the WAGO Argonautes to effect endo-RNAi silencing. Paralogous EGO-1 complexes may be involved in this and other RNAi pathways. Some of the ERIC components were omitted from the model for clarity. Change history * Abstract * Change history * Author information * Supplementary informationCorrected online 09 January 2012In the version of this article initially published, information in Table 1 was inaccurate. "Newly described" should have been "novel" and "Argonaute protein domain" should have read "Argonaute protein." The errors have been corrected in the HTML and PDF versions of the article. Author information * Abstract * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Caroline Thivierge & * Neetha Makil Affiliations * Department of Biochemistry, McGill University, Montreal, Quebec, Canada. * Caroline Thivierge, * Neetha Makil, * Mathieu Flamand & * Thomas F Duchaine * Goodman Cancer Center, McGill University, Montreal, Quebec, Canada. * Caroline Thivierge, * Neetha Makil, * Mathieu Flamand & * Thomas F Duchaine * Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Jessica J Vasale, * Craig C Mello & * Darryl Conte Jr * Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Craig C Mello * Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA. * James Wohlschlegel Contributions C.T. conducted the experiments presented in Figures 1c, 2c,d, 3a,b, 4a,b,d and 5a, prepared the figures and assisted with the preparation of the manuscript. N.M. conducted the experiments presented in Figure 1a,d, the ERI-5 samples in 1e and 2a,b. M.F. conducted the experiments presented in Figure 4b,d, and assisted with the model. J.W. carried out the MuDPIT analyses of IP samples. D.C. and J.J.V. conducted the experiments in Figure 3c, under C.C.M.'s direction. D.C. provided scientific advice, and assisted with the redaction of the manuscript. T.F.D. conducted the experiments in Figure 1b, wrote the manuscript and directed the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Thomas F Duchaine Author Details * Caroline Thivierge Search for this author in: * NPG journals * PubMed * Google Scholar * Neetha Makil Search for this author in: * NPG journals * PubMed * Google Scholar * Mathieu Flamand Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica J Vasale Search for this author in: * NPG journals * PubMed * Google Scholar * Craig C Mello Search for this author in: * NPG journals * PubMed * Google Scholar * James Wohlschlegel Search for this author in: * NPG journals * PubMed * Google Scholar * Darryl Conte Jr Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas F Duchaine Contact Thomas F Duchaine Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–4, Supplementary Results and Supplementary Methods Excel files * Supplementary Data 1 (123K) Supplementary data of the coding loci targeted by 26G-RNAs in eri-5 and rrf-3 embryos. * Supplementary Data 2 (2M) Complement to Supplementary Figure 3: small RNA defect of eri-5 mutant. Additional data Entities in this article * Enhanced RNAi (RNA interference) protein 5 eri-5 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Endoribonuclease dcr-1 dcr-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Enhancer of Ksr-1 Lethality ekl-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * RNAi DEfective family member (rde-4) rde-4 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Dicer related helicase protein 1 drh-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * RNAi DEfective family member (rde-1) rde-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * RNA-dependent RNA polymerase family member (rrf-1) rrf-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * RNA-directed RNA polymerase related EGO-1 ego-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * ALG-3 T22B3.2 Caenorhabditis elegans * View in Entrez Gene * ALG-4 tag-76 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Endogenous-RNAi deficient argonaute protein 1 ergo-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * RNA-dependent RNA polymerase family member (rrf-3) rrf-3 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Dicer Related Helicase family member (drh-3) drh-3 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * 3'-5' exonuclease eri-1 eri-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Enhanced RNAi (RNA interference) protein 3 eri-3 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Enhanced RNAi (RNA interference) protein 9 C26E6.7 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Phosphatase Interacting with RNA/RNP family member (pir-1) pir-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Argonaute (plant)-Like Gene family member (alg-1) alg-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Argonaute (plant)-Like Gene alg-2 Caenorhabditis elegans * View in Entrez Gene * Protein T06A10.3 Caenorhabditis elegans * View in UniProt * Protein B0001.2 B0001.2 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Ribonuclease 3 DROSHA Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Microprocessor complex subunit DGCR8 DGCR8 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * Dicer-2 Dcr-2 Drosophila melanogaster * View in UniProt * View in Entrez Gene * R2D2 r2d2 Drosophila melanogaster * View in UniProt * View in Entrez Gene * Protein C40A11.10 C40A11.10 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Suppressor/Enhancer of Lin-12 family member (sel-1) sel-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Protein Dicer dcr1 Schizosaccharomyces pombe (strain 972 / ATCC 24843) * View in UniProt * View in Entrez Gene * Endoribonuclease Dicer DICER1 Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia - A cis-antisense RNA acts in trans in Staphylococcus aureus to control translation of a human cytolytic peptide
- Nat Struct Mol Biol 19(1):105-112 (2012)
Nature Structural & Molecular Biology | Article Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H * Ying Shen1 * Kyung Duk Koh1 * Bernard Weiss2 * Francesca Storici1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:98–104Year published:(2012)DOI:doi:10.1038/nsmb.2176Received 21 December 2010 Accepted 16 September 2011 Published online 04 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Numerous studies have shown that ribonucleoside monophosphates (rNMPs) are probably abundant among all nonstandard nucleotides occurring in genomic DNA. Therefore, it is important to understand to what extent rNMPs may alter genome integrity and what factors affect their stability. We developed oligonucleotide-driven gene correction assays in Escherichia coli and Saccharomyces cerevisiae to show that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis and produce a genetic change. We discovered that isolated mispaired rNMPs in chromosomal DNA are removed by the mismatch repair system in competition with RNase H type 2. However, a mismatch within an RNA-DNA heteroduplex region requires RNase H type 1 for removal. In the absence of mismatch repair and RNases H, ribonucleotide-driven gene modification increased by a factor of 47 in yeast and 77,000 in E. coli. View full text Figures at a glance * Figure 1: Diagrams and sequences of the loci targeted by the RNA-containing oligonucleotides. () The lacZ locus containing a two-base deletion and a substitution mutation targeted by the LacZ.R6I2, LacZ.R2.47I2, LacZ.R1S1 or LacZ.R5S1 oligonucleotide. () The lacZ locus containing a substitution mutation targeted by the LacZ.R1S1 or LacZ.R5S1 oligonucleotide. () The rpsL locus targeted by the RpsL.R1S1 oligonucleotide. (–) The trp5 locus containing a two-base deletion and substitution mutations targeted by the TRP5.R2_R1I2_S1 oligonucleotide (), containing just a substitution mutation targeted by the TRP5.R1S1 oligonucleotide () or containing only a two-base deletion mutation targeted by the TRP5.R2I2 oligonucleotide (). In the name of the RNA-containing oligonucleotides, substitutions are indicated by a subscript capital 'S' and insertions by a subscript capital 'I'. The letters 'S' and 'I' are followed by a subscript number indicating the number of bases that are substituted or inserted, respectively. * Figure 2: RNase HII cleavage specificity. () Structural presentation of 5′-radiolabeled (32P, indicated by a purple asterisks) substrates (S1–S11) and cleavage percentage for each substrate, expressed as median and range (in parentheses) from three independent samples. Inverted triangles indicate the cleavage sites. () Denaturing polyacrylamide gels showing fragments resulting from cleavage using RNase HII. M, 20- to 100-nt oligonucleotide marker. The gel images were cropped above the 50-nt band of the marker. S1–S11, substrates used; nt, nucleotide. () Substrates used in the experiment shown in panel and their cleavage percentage, expressed as mean and range (in parentheses) from two independent samples. () Denaturing polyacrylamide gel showing fragments resulting from cleavage using reduced amount of RNase HII and shorter incubation time. M, 20- to 100-nt oligonucleotide marker. The gel image was cropped as in . S2, S4, S6, S11 are substrates used. Author information * Abstract * Author information * Supplementary information Affiliations * School of Biology, Georgia Institute of Technology, Atlanta, Georgia, USA. * Ying Shen, * Kyung Duk Koh & * Francesca Storici * Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. * Bernard Weiss Contributions Y.S. conducted most of the experiments on E. coli, all yeast experiments and statistical analyses of the data. K.D.K. carried out the RNase HII cleavage experiments, analyzed biochemical data and helped with the E. coli experiments. B.W. helped to design the experiments, conducted initial tests on E. coli and analyzed data. F.S. designed most of experiments, analyzed data and wrote the manuscript, with input from all authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Francesca Storici Author Details * Ying Shen Search for this author in: * NPG journals * PubMed * Google Scholar * Kyung Duk Koh Search for this author in: * NPG journals * PubMed * Google Scholar * Bernard Weiss Search for this author in: * NPG journals * PubMed * Google Scholar * Francesca Storici Contact Francesca Storici Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–2 and Supplementary Tables 1–6 Additional data Entities in this article * DNA topoisomerase 1 TOP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA mismatch repair protein mutS mutS Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Beta-galactosidase lacZ Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Ribonuclease HI rnhA Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Ribonuclease HII rnhB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * 30S ribosomal protein S12 rpsL Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Tryptophan synthase TRP5 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA mismatch repair protein MSH2 MSH2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ribonuclease H2 subunit A RNH201 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA polymerase epsilon catalytic subunit A POL2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA mismatch repair protein MSH6 MSH6 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA-directed DNA/RNA polymerase mu POLM Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * DNA polymerase beta POLB Homo sapiens * View in UniProt * View in Entrez Gene * View in Antibodypedia * DNA polymerase III subunit epsilon dnaQ Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA polymerase III subunit alpha dnaE Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * 3-isopropylmalate dehydrogenase LEU2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - Single-molecule studies reveal the function of a third polymerase in the replisome
- Nat Struct Mol Biol 19(1):113-116 (2012)
Nature Structural & Molecular Biology | Article A cis-antisense RNA acts in trans in Staphylococcus aureus to control translation of a human cytolytic peptide * Nour Sayed1 * Ambre Jousselin1 * Brice Felden1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:105–112Year published:(2012)DOI:doi:10.1038/nsmb.2193Received 09 July 2011 Accepted 31 October 2011 Published online 25 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Antisense RNAs (asRNAs) pair to RNAs expressed from the complementary strand, and their functions are thought to depend on nucleotide overlap with genes on the opposite strand. There is little information on the roles and mechanisms of asRNAs. We show that a cis asRNA acts in trans, using a domain outside its target complementary sequence. SprA1 small regulatory RNA (sRNA) and SprA1AS asRNA are concomitantly expressed in S. aureus. SprA1AS forms a complex with SprA1, preventing translation of the SprA1-encoded open reading frame by occluding translation initiation signals through pairing interactions. The SprA1 peptide sequence is within two RNA pseudoknots. SprA1AS represses production of the SprA1-encoded cytolytic peptide in trans, as its overlapping region is dispensable for regulation. These findings demonstrate that sometimes asRNA functional domains are not their gene-target complementary sequences, suggesting there is a need for mechanistic re-evaluation of asRNAs ex! pressed in prokaryotes and eukaryotes. View full text Figures at a glance * Figure 1: Genomic location, lengths, boundaries and expression of sprA1 and sprA1AS. () Location of sprA1-sprA1AS in S. aureus pathogenicity island SaPIn3 of the S. aureus strain Newman (NWMN) genome. () Right panels: northern-blot detection of SprA1 and SprA1AS in a wild-type Newman strain (lane 1) and in an isogenic sprA1-sprA1AS double deletion strain (lane 2). Left panels: length evaluation of SprA1 and SprA1AS adjoining synthetic labeled RNAs of known lengths combined to 5′ end determinations by RACE mapping. The nucleotide numberings of the SprA1 and SprA1AS ends refer to positions in the S. aureus Newman genomic sequence12. (,) SprA1 and SprA1AS expression profiles during S. aureus growth. The expression levels of SprA1 and SprA1AS during a 10-h growth of S. aureus Newman strain detected by northern blots. For loading controls, the blots were also probed for tmRNA. The growth curves of the Newman strains are presented, with the quantification of SprA1 (black triangles) and SprA1AS (gray diamonds) levels relative to the amount of tmRNA from the same ! RNA extraction. AU, arbitrary units. () Determination of the in vivo concentrations of SprA1 and SprA1AS in a wild-type S. aureus Newman strain during growth detected by northern blots. The quantification of SprA1 and SprA1ASin vivo levels (left panels) was carried out relative to increasing amounts of synthetic, gel-purified SprA1 and SprA1AS RNAs (the two right panels). In vivo, the ratios between the SprA1 and SprA1AS RNAs are 1:35, 1:92, 1:63 and 1:50 at A600nm levels of 4, 7, 10 and 12, respectively. * Figure 2: Detection of the interaction between SprA1 and SprA1ASin vivo and assessment of their binding constants. () Northern blot analysis of SprA1 (wild-type or tagged with a StreptoTag (ST) expression at mid-exponential (A600nm = 3) and stationary (A600nm = 11) phases in wild-type Newman (lane 3), isogenic Newman ΔsprA1-sprA1AS deletion mutant (lane 2) and Newman ΔsprA1-ΔsprA1AS pCN35Ω STsprA1-sprA1AS strain (lane 1). () Northern blot analysis of the affinity purification fractions from either Newman ΔsprA1-ΔsprA1AS pCN35ΩSTsprA1-sprA1AS extracts, or Newman wild-type pCN35ΩsprA1AS extracts, as a negative control. Labeled DNA probes were used for SprA1 (WT and tagged), for SprA1AS and for tmRNA used as an internal negative control. FT, flow through; W4, wash 4, W5, wash 5; E, elution. (,) Complex formation between purified SprA1 and SprA1AS by native gel retardation assays. Purified, labeled (asterisks) SprA1AS () or SprA1 () with increasing amounts of unlabeled SprA1 () or unlabeled SprA1AS (). The diamonds indicate the molar ratios used to perform the competition assays with! a 1,000-fold molar excess of yeast (Saccharomyces cerevisiae) total tRNAs or with a 20-fold molar excess of the indicated unlabeled RNA. The apparent binding constant between SprA1AS and SprA1 was inferred from these data: Kd = 15 ± 5 nM. * Figure 3: Experimental and phylogenetic evidence for the pairings between SprA1AS and SprA1. () Proposed pairings between SprA1 and SprA1AS. Shine-Dalgarno and 5′-GUG-3′ or 5′-AUG-3′ start codons are in red, and the SprA1AS and SprA1 interacting domains are boxed in red. Blue minus signs indicate the disappearance of the cleavages triggered by the structural probes in the RNA duplex. Triangles are the V1 cuts, arrows capped by circles are the S1 cuts, and uncapped arrows are the lead cleavages. The intensity of the cleavages is proportional to the darkness of the symbols. The blue S1 cut appears when the duplex forms. () Phylogenetic support for the proposed interaction between SprA1 and SprA1AS, when comparing the sequences of the two RNAs located in genomes and plasmids. Covariations are shown in gray, Shine-Dalgarno and start codons are boxed. (,) Experimentally supported structure of SprA1 and SprA1AS, emphasizing the 3′ overlapping sequence (yellow) as well as the experimentally and phylogenetically supported interaction region (red box). Covariations! are shown in gray. The other symbols are similar to those in panel . Structural changes detected upon complex formation are indicated in blue. The domains of the RNAs are indicated (SprA1: H1–H6, H1-H2 junction, L1–L6, PK1-PK2; SprA1AS: H1-H2AS, H1AS-H2AS junction and L1AS-L2AS) (see also Supplementary Figs. 3–5). * Figure 4: SprA1 and SprA1AS interact by their 5′ non-overlapping domains. Complex formation between labeled SprA1AS with increasing amounts of unlabeled 5′ SprA1 () or 3′ SprA1 () and between labeled SprA1 with increasing amounts of unlabeled 5′ SprA1AS () or 3′ SprA1AS (), as detected by native gel retardation assays. The apparent binding constants between the RNAs were inferred from these data. For SprA1AS–5′ SprA1, the Kd is 16 ± 5 nM. For SprA1–5′ SprA1AS, the Kd is 300 ± 50 nM. There is no duplex formation between SprA1 and 3′ SprA1AS or between 3′ SprA1 and SprA1AS. The black diamonds indicate the molar ratios used to carry out the competition assays with a 2,000-fold molar excess of poly(U) RNAs or with a 20-fold molar excess of the indicated unlabeled RNA. Asterisks indicate the 32P-radiolabeled RNAs (see also Supplementary Fig. 6). * Figure 5: SprA1 recruits the S. aureus ribosomes and is translated in vitro, and SprA1AS hinders SprA1 translation by its 5′ non-overlapping domain. () SprA1 structure indicating the ribosome toeprints (oval), the reverse transcriptase (RT) pause in the presence of sprA1AS (black arrows) and the mutated nucleotides in the SD–mutated SprA1 construct (rectangles, 12 mutated nucleotides to maintain H1 while modifying the Shine-Dalgarno sequence). The predicted initiation and termination codons are framed, and the nucleotide sequence overlapping with sprA1AS is in gray. () S. aureus ribosome toeprint assay of SprA1 (WT SprA1) and the disappearance of the toeprints in the SD-mutated SprA1. In the presence of SprA1AS at a 2:1 molar ratio, there are no toeprints, indicating that the asRNA impairs ribosome loading onto SprA1. The toeprints are indicated with a black bullet and the reverse transcriptase pause for SprA1, in the presence of SprA1AS, is indicated by an arrowhead. T, A, G and C are the SprA1 sequencing ladders. () In vitro translation of SprA1 (lane 1), of SprA1 in the presence of SprA1AS at a 1:1 molar ratio (lane! 2), of SprA1 in the presence of 5′ SprA1AS at a 1:10 molar ratio (lane 3), of SprA1 in the presence of 3′ SprA1AS at a 1:10 molar ratio (lane 4) and of SD-mutated SprA1 (lane 5). The translated SprA1-encoded polypeptide of ~3 kDa is indicated by an arrowhead. () Northern blot analysis of SprA1 and SprA1AS in Newman pCN35 and isogenic Newman pCN35ΩsprA1AS during growth. The 5S rRNAs are the controls. * Figure 6: SprA1AScis-RNA acts in trans to downregulate SprA1-encoded peptide expression in vivo. () Detection of the ~5 kDa SprA1-encoded flagged peptide at early (A600nm = 1) and mid-exponential (A600nm = 5) phases of growth in strains Newman ΔsprA1-ΔsprA1AS pCN34ΩsprA1tag pCN35 (lanes 1 and 3) and in isogenic Newman ΔsprA1-ΔsprA1AS pCN34ΩsprA1tag pCN35ΩsprA1AS strain (lanes 2 and 4) by immunoblots using anti-Flag antibodies. () Northern blot analysis for monitoring SprA1-Flag RNA (upper panel) and SprA1AS RNA (lower panel) expression levels at identical phases of growth. The 5S rRNAs are the internal loading controls. * Figure 7: The SprA1-encoded peptide is lytic for human cells. () Hemolytic activity of synthetic SprA1-encoded peptide compared to a non-hemolytic peptide used as a negative control. Controls: the minus sign indicates that PBS was added to the red blood cells (RBC), the plus sign indicates hypotonic solution was added to RBCs. RBC sedimentation indicates the absence of hemolysis, whereas a red supernatant implies hemolysis. () Differential hemolytic activity of the synthetic SprA1 peptide for human and sheep RBCs. The peptide induces a strong hemolysis on the human RBCs but a weak hemolysis on the sheep RBCs. () Proposed model for the downregulation of SprA1 sRNA internal translation in trans by the cis-encoded SprA1AS. The SprA1 internal ORF is shown in green and the SprA1 and SprA1AS 5′ non-overlapping interacting domains are in red. Their 3′ overlapping domains are in yellow. Upon duplex formation, the SprA1AS 5′ domain pairs at and around the SprA1 internal translation initiation signals (SD-sequence and start codon, red) by ! unfolding pseudoknot PK1. During S. aureus growth, translation of the SprA1-encoded peptide is repressed by base pairings in trans with SprA1AS RNA (see also Supplementary Fig. 7). Author information * Abstract * Author information * Supplementary information Affiliations * Laboratoire de Biochimie Pharmaceutique Inserm U835 Upres EA2311 Université de Rennes, Rennes, France. * Nour Sayed, * Ambre Jousselin & * Brice Felden Contributions N.S. and B.F. designed experiments, prepared samples, analyzed the data and wrote the manuscript. A.J. constructed the sRNA double mutant, did the Hfq experiment and participated in discussions and writing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Brice Felden Author Details * Nour Sayed Search for this author in: * NPG journals * PubMed * Google Scholar * Ambre Jousselin Search for this author in: * NPG journals * PubMed * Google Scholar * Brice Felden Contact Brice Felden Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7 and Supplementary Tables 1–2 Additional data Entities in this article * Beta-lactamase blaZ Staphylococcus aureus * View in UniProt * View in Entrez Gene * RNA chaperone, host factor-1 protein NWMN_1212 Staphylococcus aureus (strain Newman) * View in UniProt * View in Entrez Gene * Delta-hemolysin Staphylococcus aureus * View in UniProt - Fluorescent fusion protein knockout mediated by anti-GFP nanobody
- Nat Struct Mol Biol 19(1):117-121 (2012)
Nature Structural & Molecular Biology | Brief Communication Single-molecule studies reveal the function of a third polymerase in the replisome * Roxana E Georgescu1 * Isabel Kurth1 * Mike E O'Donnell1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:113–116Year published:(2012)DOI:doi:10.1038/nsmb.2179Received 16 June 2011 Accepted 29 September 2011 Published online 11 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The Escherichia coli replisome contains three polymerases, one more than necessary to duplicate the two parental strands. Using single-molecule studies, we reveal two advantages conferred by the third polymerase. First, dipolymerase replisomes are inefficient at synthesizing lagging strands, leaving single-strand gaps, whereas tripolymerase replisomes fill strands almost to completion. Second, tripolymerase replisomes are much more processive than dipolymerase replisomes. These features account for the unexpected three-polymerase-structure of bacterial replisomes. View full text Figures at a glance * Figure 1: TriPol replisomes are more processive than DiPol replisomes. () Scheme of single-molecule experiments. For clarity, only the DiPol replisome is illustrated. () DNA products from either the DiPol (left) or TriPol (right) replisome, using 250 nM primase. The endpoints of two representative DNA products are marked with arrowheads. () DNA length distribution histograms. Numbers represent the single-exponential fit ± s.e.m. of the total number (N) of molecules analyzed. Gray bars represent DNA strand lengths below 15 kb that were undersampled because they were obscured by the width of the diffusion barrier. Left, DiPol replisomes, right, TriPol replisomes. () Processivity of DiPol and TriPol replisomes, where the indicated polymerase is present or absent from the buffer flow. * Figure 2: TriPol replisomes are more efficient on the lagging strand than DiPol replisomes are. () Scheme of the bead-based assay; the DiPol replisome is illustrated for simplicity. () Dipol and Tripol replisomes replicate DNA with similar rates. Left, autoradiogram of 0.8% alkaline agarose gel analysis of reactions, using either Tripol III* (20 nM) or DiPol III* (80 nM); DnaG primase concentration was 200 nM. Right, plot of DNA length versus time. () Left, leading- and lagging-strand replication products from bead-based reactions, resolved on denaturing agarose gels, using 320 nM DnaG primase. Right, quantitation of leading- and lagging-strand synthesis, normalized to the products of the TriPol replisome. * Figure 3: Analysis of ssDNA gaps in lagging strand products. () Magnified view of DNA products generated by DiPol and TriPol replisomes; the light and dark regions correspond to dsDNA segments and ssDNA gaps. () Comparative histogram showing the percentage of DNA strands with gaps (green) and without gaps (purple). () Histograms showing the distribution of gap length (in μm) using DiPol (red) and TriPol (blue) replisomes. () Model of TriPol and DiPol replisome action. Pol III cores are represented as right hands; with the β-clamp (red), clamp loader (dark green), DnaB helicase (blue hexamer), primase (light green) and SSB (purple). The τ-subunit C-terminal domains (IV and V) are illustrated as jointed lines that mediate connections to DnaB helicase and Pol III cores. The χψψ subunits of the clamp loader are omitted for clarity. The TriPol replisome depicts two Pol III cores extending two Okazaki fragments simultaneously, although there are other ways a TriPol replisome can be used (see text). The left illustration depicts one la! gging Pol III extending an RNA primer (red) to produce a DNA strand (yellow), and the other lagging Pol III core extends the DNA (blue) to fill a ssDNA gap. Author information * Author information * Supplementary information Affiliations * The Rockefeller University, Howard Hughes Medical Institute, New York, New York, USA. * Roxana E Georgescu, * Isabel Kurth & * Mike E O'Donnell Contributions R.E.G. and I.K. carried out experiments; R.E.G., I.K. and M.E.O. designed the experiments. R.E.G., I.K. and M.E.O. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mike E O'Donnell Author Details * Roxana E Georgescu Search for this author in: * NPG journals * PubMed * Google Scholar * Isabel Kurth Search for this author in: * NPG journals * PubMed * Google Scholar * Mike E O'Donnell Contact Mike E O'Donnell Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (324K) Supplementary Figures 1–4 and Supplementary Methods Movies * Supplementary Video 1 (1M) Replication performed by Dipol replisomes. An example of a movie depicting real-time observation of coupled leading/lagging strand replication of a mini-rolling circle substrate by E. coli Dipol replisomes. The force of the hydrodynamic flow pushes the DNA-lipid complex to a diffusion barrier etched in the glass surface and concentrates numerous DNA molecules in the visual field shown here. The width of the visible area in the direction of the flow is 73 μm (equivalent to 220 kb) and the flow direction is from top to the bottom. Individual DNA molecules visualized with the fluorescent dye Yo-Pro1 are stretched by the buffer flow (100 μl/min) and imaged through Total Internal Reflection Fluorescence (TIRF) microscopy. Toward the end of the movie, the buffer-flow is stopped, letting the strands recoil, then the buffer flow is started again. Movie contains circa 7' 30″ of experimental data rendered at 20 frames per second (original data acquisition is 1 frame/s at 100 ms ex! posure per frame). * Supplementary Video 2 (332K) Replication performed by Tripol replisomes. The video depicts the recording of a replication reaction performed by Tripol replisomes. The movie contains circa 5' 30″ of experimental data rendered at 20 frames per second (original data acquisition is 1 frame/s at 100 ms exposure for each frame). * Supplementary Video 3 (451K) DNA molecules that harbor duplex regions contain gaps on the same molecule. The video depicts a recording at the end of a replication reaction using a DiPol replisome. The flow of the buffer solution is stopped then restarted, allowing the DNA strands to stretch and then recoil to their point of origin. * Supplementary Video 4 (2M) Use of fluorescent SSB to identify ssDNA in DNA products. The video depicts three successive recordings of different DNA products of DiPol replisomes, in which reactions contained fluorescently labeled SSB. The three successive recordings are easy to identify since they have different dimensions. The videos show that DNA products contain fluorescently labeled E. coli SSB (with Oregon Green488 Maleimide). The duplex DNA is not visualized because Yo-Pro1 is omitted from the buffer flow for these experiments. To distinguish SSB bound to DNA from SSB that binds non-specifically to the surface of the flow cell, the buffer-flow is alternatively stopped and restarted in order to observe the recoiling of the DNA strands. Fluorescent SSB bound to DNA recoils and re-extends in synchrony with the changes in buffer flow (while non-specifically bound SSB does not change position). Additional data Entities in this article * DNA polymerase III subunit tau dnaX Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Replicative DNA helicase dnaB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Single-stranded DNA-binding protein ssb Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA polymerase I polA Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA polymerase II polB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA polymerase IV dinB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA primase dnaG Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene - A metal switch for controlling the activity of molecular motor proteins
- Nat Struct Mol Biol 19(1):122-127 (2012)
Nature Structural & Molecular Biology | Brief Communication Single-molecule studies reveal the function of a third polymerase in the replisome * Roxana E Georgescu1 * Isabel Kurth1 * Mike E O'Donnell1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 19,Pages:113–116Year published:(2012)DOI:doi:10.1038/nsmb.2179Received 16 June 2011 Accepted 29 September 2011 Published online 11 December 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The Escherichia coli replisome contains three polymerases, one more than necessary to duplicate the two parental strands. Using single-molecule studies, we reveal two advantages conferred by the third polymerase. First, dipolymerase replisomes are inefficient at synthesizing lagging strands, leaving single-strand gaps, whereas tripolymerase replisomes fill strands almost to completion. Second, tripolymerase replisomes are much more processive than dipolymerase replisomes. These features account for the unexpected three-polymerase-structure of bacterial replisomes. View full text Figures at a glance * Figure 1: TriPol replisomes are more processive than DiPol replisomes. () Scheme of single-molecule experiments. For clarity, only the DiPol replisome is illustrated. () DNA products from either the DiPol (left) or TriPol (right) replisome, using 250 nM primase. The endpoints of two representative DNA products are marked with arrowheads. () DNA length distribution histograms. Numbers represent the single-exponential fit ± s.e.m. of the total number (N) of molecules analyzed. Gray bars represent DNA strand lengths below 15 kb that were undersampled because they were obscured by the width of the diffusion barrier. Left, DiPol replisomes, right, TriPol replisomes. () Processivity of DiPol and TriPol replisomes, where the indicated polymerase is present or absent from the buffer flow. * Figure 2: TriPol replisomes are more efficient on the lagging strand than DiPol replisomes are. () Scheme of the bead-based assay; the DiPol replisome is illustrated for simplicity. () Dipol and Tripol replisomes replicate DNA with similar rates. Left, autoradiogram of 0.8% alkaline agarose gel analysis of reactions, using either Tripol III* (20 nM) or DiPol III* (80 nM); DnaG primase concentration was 200 nM. Right, plot of DNA length versus time. () Left, leading- and lagging-strand replication products from bead-based reactions, resolved on denaturing agarose gels, using 320 nM DnaG primase. Right, quantitation of leading- and lagging-strand synthesis, normalized to the products of the TriPol replisome. * Figure 3: Analysis of ssDNA gaps in lagging strand products. () Magnified view of DNA products generated by DiPol and TriPol replisomes; the light and dark regions correspond to dsDNA segments and ssDNA gaps. () Comparative histogram showing the percentage of DNA strands with gaps (green) and without gaps (purple). () Histograms showing the distribution of gap length (in μm) using DiPol (red) and TriPol (blue) replisomes. () Model of TriPol and DiPol replisome action. Pol III cores are represented as right hands; with the β-clamp (red), clamp loader (dark green), DnaB helicase (blue hexamer), primase (light green) and SSB (purple). The τ-subunit C-terminal domains (IV and V) are illustrated as jointed lines that mediate connections to DnaB helicase and Pol III cores. The χψψ subunits of the clamp loader are omitted for clarity. The TriPol replisome depicts two Pol III cores extending two Okazaki fragments simultaneously, although there are other ways a TriPol replisome can be used (see text). The left illustration depicts one la! gging Pol III extending an RNA primer (red) to produce a DNA strand (yellow), and the other lagging Pol III core extends the DNA (blue) to fill a ssDNA gap. Author information * Author information * Supplementary information Affiliations * The Rockefeller University, Howard Hughes Medical Institute, New York, New York, USA. * Roxana E Georgescu, * Isabel Kurth & * Mike E O'Donnell Contributions R.E.G. and I.K. carried out experiments; R.E.G., I.K. and M.E.O. designed the experiments. R.E.G., I.K. and M.E.O. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mike E O'Donnell Author Details * Roxana E Georgescu Search for this author in: * NPG journals * PubMed * Google Scholar * Isabel Kurth Search for this author in: * NPG journals * PubMed * Google Scholar * Mike E O'Donnell Contact Mike E O'Donnell Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (324K) Supplementary Figures 1–4 and Supplementary Methods Movies * Supplementary Video 1 (1M) Replication performed by Dipol replisomes. An example of a movie depicting real-time observation of coupled leading/lagging strand replication of a mini-rolling circle substrate by E. coli Dipol replisomes. The force of the hydrodynamic flow pushes the DNA-lipid complex to a diffusion barrier etched in the glass surface and concentrates numerous DNA molecules in the visual field shown here. The width of the visible area in the direction of the flow is 73 μm (equivalent to 220 kb) and the flow direction is from top to the bottom. Individual DNA molecules visualized with the fluorescent dye Yo-Pro1 are stretched by the buffer flow (100 μl/min) and imaged through Total Internal Reflection Fluorescence (TIRF) microscopy. Toward the end of the movie, the buffer-flow is stopped, letting the strands recoil, then the buffer flow is started again. Movie contains circa 7' 30″ of experimental data rendered at 20 frames per second (original data acquisition is 1 frame/s at 100 ms ex! posure per frame). * Supplementary Video 2 (332K) Replication performed by Tripol replisomes. The video depicts the recording of a replication reaction performed by Tripol replisomes. The movie contains circa 5' 30″ of experimental data rendered at 20 frames per second (original data acquisition is 1 frame/s at 100 ms exposure for each frame). * Supplementary Video 3 (451K) DNA molecules that harbor duplex regions contain gaps on the same molecule. The video depicts a recording at the end of a replication reaction using a DiPol replisome. The flow of the buffer solution is stopped then restarted, allowing the DNA strands to stretch and then recoil to their point of origin. * Supplementary Video 4 (2M) Use of fluorescent SSB to identify ssDNA in DNA products. The video depicts three successive recordings of different DNA products of DiPol replisomes, in which reactions contained fluorescently labeled SSB. The three successive recordings are easy to identify since they have different dimensions. The videos show that DNA products contain fluorescently labeled E. coli SSB (with Oregon Green488 Maleimide). The duplex DNA is not visualized because Yo-Pro1 is omitted from the buffer flow for these experiments. To distinguish SSB bound to DNA from SSB that binds non-specifically to the surface of the flow cell, the buffer-flow is alternatively stopped and restarted in order to observe the recoiling of the DNA strands. Fluorescent SSB bound to DNA recoils and re-extends in synchrony with the changes in buffer flow (while non-specifically bound SSB does not change position). Additional data Entities in this article * DNA polymerase III subunit tau dnaX Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Replicative DNA helicase dnaB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Single-stranded DNA-binding protein ssb Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA polymerase I polA Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA polymerase II polB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA polymerase IV dinB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * DNA primase dnaG Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene
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