Latest Articles Include:
- Helicase Multitasking in Ribosome Assembly
Guenther UP Jankowsky E - Mol Cell 36(4):537-538 (2009)
In this issue of Molecular Cell, Bohnsack et al. (2009) identify multiple binding sites of the RNA helicase Prp43 on preribosomal RNA. The target regions suggest distinct functions of Prp43 in ribosome biogenesis. - Fungal BRCA2 Ortholog Brh2 Brings 5′ End Strand Invasion Back on Stage
Llorente B Modesti M - Mol Cell 36(4):539-540 (2009)
In this issue of Molecular Cell, Mazloum and Holloman (2009b) propose that 5′ end strand invasion promoted by collaboration between Rad51 and Brh2 could be used for bypassing lesions during DNA replication, potentially advancing understanding of BRCA2 tumor suppressor function. - Progression through the RNA Polymerase II CTD Cycle
Buratowski S - Mol Cell 36(4):541-546 (2009)
The C-terminal domain of RNA polymerase II's largest subunit undergoes dynamic phosphorylation during transcription, and the different phosphorylation patterns that predominate at each stage of transcription recruit the appropriate set of mRNA-processing and histone-modifying factors. Recent papers help to explain how the changes in CTD phosphorylation pattern are linked to the progression from initiation through elongation to termination. - EGF-Induced ERK Activation Promotes CK2-Mediated Disassociation of α-Catenin from β-Catenin and Transactivation of β-Catenin
Ji H Wang J Nika H Hawke D Keezer S Ge Q Fang B Fang X Fang D Litchfield DW Aldape K Lu Z - Mol Cell 36(4):547-559 (2009)
Increased transcriptional activity of β-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation remains unclear. We demonstrate here that EGFR activation results in disruption of the complex of β-catenin and α-catenin, thereby abrogating the inhibitory effect of α-catenin on β-catenin transactivation via CK2α-dependent phosphorylation of α-catenin at S641. ERK2, which is activated by EGFR signaling, directly binds to CK2α via the ERK2 docking groove and phosphorylates CK2α primarily at T360/S362, subsequently enhancing CK2α activity toward α-catenin phosphorylation. In addition, levels of α-catenin S641 phosphorylation correlate with levels of ERK1/2 activity in human glioblastoma specimens and with grades of glioma malignancy. This EGFR-ERK-CK2-mediated phosphorylation of α-catenin promotes β-catenin transactivation and tumor cell i! nvasion. These findings highlight the importance of the crosstalk between EGFR and Wnt pathways in tumor development. - An Autoinhibitory Tyrosine Motif in the Cell-Cycle-Regulated Nek7 Kinase Is Released through Binding of Nek9
Richards MW O'Regan L Mas-Droux C Blot JM Cheung J Hoelder S Fry AM Bayliss R - Mol Cell 36(4):560-570 (2009)
Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the αC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selec! tive kinase inhibitors. - The SXL-UNR Corepressor Complex Uses a PABP-Mediated Mechanism to Inhibit Ribosome Recruitment to msl-2 mRNA
Duncan KE Strein C Hentze MW - Mol Cell 36(4):571-582 (2009)
Drosophila female viability requires translational repression of msl-2 mRNA by the SXL-UNR 3′ UTR corepressor complex, which inhibits ribosome recruitment by an unknown mechanism. Here, we reveal a key role for the poly(A)-binding protein (PABP), a translational activator, in this inhibitory mechanism. Efficient msl-2 mRNA silencing via the 3′ UTR requires both a poly(A) tail and PABP function, and we find that UNR directly interacts with PABP. To investigate how the repressor complex and PABP affect RNP composition during early steps in translation initiation, we established direct biochemical assays for synergistic recruitment of eIF4F and ribosomes by the cap and poly(A) tail. We find that the repressor complex targets ribosome binding after PABP-mediated recruitment of eIF4E/G. Our results uncover an important regulatory mechanism of Drosophila dosage compensation and provide insight into PABP-dependent translational control by 3′ UTR-bound regulatory protein! s. - Prp43 Bound at Different Sites on the Pre-rRNA Performs Distinct Functions in Ribosome Synthesis
Bohnsack MT Martin R Granneman S Ruprecht M Schleiff E Tollervey D - Mol Cell 36(4):583-592 (2009)
Yeast ribosome synthesis requires 19 different RNA helicases, but none of their pre-rRNA-binding sites were previously known, making their precise functions difficult to determine. Here we identify multiple binding sites for the helicase Prp43 in the 18S and 25S rRNA regions of pre-rRNAs, using UV crosslinking. Binding in 18S was predominantly within helix 44, close to the site of 18S 3′ cleavage, in which Prp43 is functionally implicated. Four major binding sites were identified in 25S, including helix 34. In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association. Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions. We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct fu! nctions at different locations. - The Evolutionarily Conserved Core Design of the Catalytic Activation Step of the Yeast Spliceosome
Fabrizio P Dannenberg J Dube P Kastner B Stark H Urlaub H Lührmann R - Mol Cell 36(4):593-608 (2009)
Metazoan spliceosomes exhibit an elaborate protein composition required for canonical and alternative splicing. Thus, the minimal set of proteins essential for activation and catalysis remains elusive. We therefore purified in vitro assembled, precatalytic spliceosomal complex B, activated Bact, and step 1 complex C from the simple eukaryote Saccharomyces cerevisiae. Mass spectrometry revealed that yeast spliceosomes contain fewer proteins than metazoans and that each functional stage is very homogeneous. Dramatic compositional changes convert B to Bact, which is composed of 40 evolutionarily conserved proteins that organize the catalytic core. Additional remodeling occurs concomitant with step 1, during which nine proteins are recruited to form complex C. The moderate number of proteins recruited to complex C will allow investigations of the chemical reactions in a fully defined system. Electron microscopy reveals high-quality images of yeast spliceosomes at defined f! unctional stages, indicating that they are well-suited for three-dimensional structure analyses. - An RPA-Related Sequence-Specific DNA-Binding Subunit of Telomerase Holoenzyme Is Required for Elongation Processivity and Telomere Maintenance
Min B Collins K - Mol Cell 36(4):609-619 (2009)
Telomerase ribonucleoprotein complexes copy an internal RNA template to synthesize DNA repeats. DNA-interacting subunits other than telomerase reverse transcriptase (TERT) and telomerase RNA (TER) have been hypothesized to account for high repeat addition processivity of telomerase holoenzyme compared to the minimal catalytic RNP. Here, we present the identification of three additional subunits of Tetrahymena thermophila telomerase holoenzyme. Each of seven telomerase proteins is required for telomere maintenance and copurifies active RNP. The catalytic core (p65-TER-TERT) is assembled with a three-protein subcomplex (p75-p45-p19) and two peripheral subunits (p82 and p50). Remarkably, only a p82-enriched subset of the total holoenzyme population is capable of high repeat addition processivity, as shown by p82 immunodepletion and add-back. The RPA-like p82 subunit binds sequence specifically to multiple telomeric repeats. These discoveries establish the existence of a t! elomerase holoenzyme processivity subunit with sequence-specific DNA binding. - Brh2 Promotes a Template-Switching Reaction Enabling Recombinational Bypass of Lesions during DNA Synthesis
Mazloum N Holloman WK - Mol Cell 36(4):620-630 (2009)
Accumulating evidence for Rad51-catalyzed DNA strand invasion during double-strand break repair features a 3′ single-stranded tail as the preferred substrate for reaction, but paradoxically, the preferred substrate in model reactions in vitro is the 5′ end. Here, we examined the Rad51-promoted 5′ end invasion reaction in the presence of Brh2, the BRCA2 family protein in Ustilago maydis. Using plasmid DNA and a homologous duplex oligonucleotide with 5′ protruding single-stranded tail as substrates, we found that Brh2 can stimulate Rad51 to promote the formation of a four-stranded complement-stabilized D loop. In this structure, the incoming recessed complementary strand of the oligonucleotide has switched partners and can now prime DNA synthesis using the recipient plasmid DNA as template, circumventing a lesion that blocks elongation when the 5′ protruding tail serves as template for fill-in synthesis. We propose that template switching promoted by Brh2 provi! des a mechanism for recombination-mediated bypass of lesions blocking synthesis during DNA replication. - AID Produces DNA Double-Strand Breaks in Non-Ig Genes and Mature B Cell Lymphomas with Reciprocal Chromosome Translocations
Robbiani DF Bunting S Feldhahn N Bothmer A Camps J Deroubaix S McBride KM Klein IA Stone G Eisenreich TR Ried T Nussenzweig A Nussenzweig MC - Mol Cell 36(4):631-641 (2009)
Cancer-initiating translocations such as those associated with lymphomas require the formation of paired DNA double-strand breaks (DSBs). Activation-induced cytidine deaminase (AID) produces widespread somatic mutation in mature B cells; however, the extent of "off-target" DSB formation and its role in translocation-associated malignancy is unknown. Here, we show that deregulated expression of AID causes widespread genome instability, which alone is insufficient to induce B cell lymphoma; transformation requires concomitant loss of the tumor suppressor p53. Mature B cell lymphomas arising as a result of deregulated AID expression are phenotypically diverse and harbor clonal reciprocal translocations involving a group of Immunoglobulin (Ig) and non-Ig genes that are direct targets of AID. This group includes miR-142, a previously unknown micro-RNA target that is translocated in human B cell malignancy. We conclude that AID produces DSBs throughout the genome, which ! can lead to lymphoma-associated chromosome translocations in mature B cells. - Two-Step Recognition of DNA Damage for Mammalian Nucleotide Excision Repair: Directional Binding of the XPC Complex and DNA Strand Scanning
Sugasawa K Akagi JI Nishi R Iwai S Hanaoka F - Mol Cell 36(4):642-653 (2009)
For mammalian nucleotide excision repair (NER), DNA lesions are recognized in at least two steps involving detection of unpaired bases by the XPC protein complex and the subsequent verification of injured bases. Although lesion verification is important to ensure high damage discrimination and the accuracy of the repair system, it has been unclear how this is accomplished. Here, we show that damage verification involves scanning of a DNA strand from the site where XPC is initially bound. Translocation by the NER machinery exhibits a 5′-to-3′ directionality, strongly suggesting involvement of the XPD helicase, a component of TFIIH. Furthermore, the initial orientation of XPC binding is crucial in that only one DNA strand is selected to search for the presence of lesions. Our results dissect the intricate molecular mechanism of NER and provide insights into a strategy for mammalian cells to survey large genomes to detect DNA damage. - Rep Provides a Second Motor at the Replisome to Promote Duplication of Protein-Bound DNA
Guy CP Atkinson J Gupta MK Mahdi AA Gwynn EJ Rudolph CJ Moon PB van Knippenberg IC Cadman CJ Dillingham MS Lloyd RG McGlynn P - Mol Cell 36(4):654-666 (2009)
Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-boun! d DNA. - Discovering Hematopoietic Mechanisms through Genome-wide Analysis of GATA Factor Chromatin Occupancy
Fujiwara T O'Geen H Keles S Blahnik K Linnemann AK Kang YA Choi K Farnham PJ Bresnick EH - Mol Cell 36(4):667-681 (2009)
GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for crossregulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2-negative autoregulatory loop. These results establish fund! amental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis. - Insights into GATA-1-Mediated Gene Activation versus Repression via Genome-wide Chromatin Occupancy Analysis
Yu M Riva L Xie H Schindler Y Moran TB Cheng Y Yu D Hardison R Weiss MJ Orkin SH Bernstein BE Fraenkel E Cantor AB - Mol Cell 36(4):682-695 (2009)
The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1-induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA-binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus nondifferentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1-bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that polycomb repressive complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1-repressed genes. These data ! provide insights into GATA-1-mediated gene regulation in vivo. - Bak Activation for Apoptosis Involves Oligomerization of Dimers via Their α6 Helices
Dewson G Kratina T Czabotar P Day CL Adams JM Kluck RM - Mol Cell 36(4):696-703 (2009)
A pivotal step toward apoptosis is oligomerization of the Bcl-2 relative Bak. We recently reported that its oligomerization initiates by insertion of an exposed BH3 domain into the groove of another Bak monomer. We now report that the resulting BH3:groove dimers can be converted to the larger oligomers that permeabilize mitochondria by an interface between α6 helices. Cysteine residues placed in α6 could be crosslinked only after apoptotic signaling. Cysteines placed at both interfaces established that the BH3:groove dimer is symmetric and that the α6:α6 interface can link these dimers into homo-oligomers containing at least 18 Bak molecules. A putative zinc-binding site in α6 was not required to form the α6:α6 interface, and its mutation in full-length Bak did not affect Bak conformation, oligomerization, or function. We conclude that α6:α6 interaction occurs during Bak oligomerization and proapoptotic function, but we find no evidence that zinc binding to th! at interface regulates apoptosis. - PCNA Is Required for Initiation of Recombination-Associated DNA Synthesis by DNA Polymerase δ
Li X Stith CM Burgers PM Heyer WD - Mol Cell 36(4):704-713 (2009)
Genetic recombination ensures proper chromosome segregation during meiosis and is essential for genome stability and tumor suppression. DNA synthesis after Rad51-mediated DNA strand invasion is a crucial step during recombination. PCNA is known as the processivity clamp for DNA polymerases. Here, we report the surprising observation that PCNA is specifically required to initiate recombination-associated DNA synthesis in the extension of the 3′ end of the invading strand in a D loop. We show using a reconstituted system of yeast Rad51, Rad54, RPA, PCNA, RFC, and DNA polymerase δ that loading of PCNA by RFC targets DNA polymerase δ to the D loop formed by Rad51 protein, allowing efficient utilization of the invading 3′ end and processive DNA synthesis. We conclude that PCNA has a specific role in the initiation of recombination-associated DNA synthesis and that DNA polymerase δ promotes recombination-associated DNA synthesis.
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