Hot off the presses! Mar 04 mol cell

The Mar 04 issue of the mol cell is now up on Pubget (About mol cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• A Role for Dormant Origins in Tumor Suppression
  - mol cell 41(5):495-496 (2011)
  In this issue of Molecular Cell, Kawabata et al. (2011) show that mice hypomorphic for the replication licensing protein Mcm4 show spontaneous DNA replication defects due to a lack of dormant origins, potentially explaining why these mice are cancer prone.
• Small Molecules, Big Effects: A Role for Chromatin-Localized Metabolite Biosynthesis in Gene Regulation
  - mol cell 41(5):497-499 (2011)
  In this issue of Molecular Cell, Katoh et al. (2011) show that the biosynthetic enzyme MATIIα binds at the HO-1 locus in mammalian cells and exhibits transcriptional corepressor activity through local production of S-adenosylmethionine, which stimulates histone methyltransferases to promote a transcriptionally repressive chromatin environment.
• PI3K p110β: More Tightly Controlled or Constitutively Active?
  - mol cell 41(5):499-501 (2011)
  In this issue of Molecular Cell, Zhang et al. (2011) report the structure of the p110β/p85β complex and identify a new inhibitory interaction between p110β and the p85 cSH2 domain. The tighter control of p110β mediated by this interaction appears to be balanced by the disruption of inhibitory contacts with the p85 iSH2 domain (Dbouk et al., 2010).
• Histone Chaperones: Modulators of Chromatin Marks
  - mol cell 41(5):502-514 (2011)
  The many factors that control chromatin biology play key roles in essential nuclear functions like transcription, DNA damage response and repair, recombination, and replication and are critical for proper cell-cycle progression, stem cell renewal, differentiation, and development. These players belong to four broad classes: histone modifiers, chromatin remodelers, histone variants, and histone chaperones. A large number of studies have established the existence of an intricate functional crosstalk between the different factors, not only within a single class but also between different classes. In light of this, while many recent reviews have focused on structure and functions of histone chaperones, the current text highlights novel and striking links that have been established between these proteins and posttranslational modifications of histones and discusses the functional consequences of this crosstalk. These findings feed a current hot question of how cell memory m! ay be maintained through epigenetic mechanisms involving histone chaperones.
• Regulation of Homologous Recombination by RNF20-Dependent H2B Ubiquitination
  - mol cell 41(5):515-528 (2011)
  The E3 ubiquitin ligase RNF20 regulates chromatin structure by monoubiquitinating histone H2B in transcription. Here, we show that RNF20 is localized to double-stranded DNA breaks (DSBs) independently of H2AX and is required for the DSB-induced H2B ubiquitination. In addition, RNF20 is required for the methylation of H3K4 at DSBs and the recruitment of the chromatin-remodeling factor SNF2h. Depletion of RNF20, depletion of SNF2h, or expression of the H2B mutant lacking the ubiquitination site (K120R) compromises resection of DNA ends and recruitment of RAD51 and BRCA1. Consequently, cells lacking RNF20 or SNF2h and cells expressing H2B K120R exhibit pronounced defects in homologous recombination repair (HRR) and enhanced sensitivity to radiation. Finally, the function of RNF20 in HRR can be partially bypassed by forced chromatin relaxation. Thus, the RNF20-mediated H2B ubiquitination at DSBs plays a critical role in HRR through chromatin remodeling.
• Requirement of ATM-Dependent Monoubiquitylation of Histone H2B for Timely Repair of DNA Double-Strand Breaks
  - mol cell 41(5):529-542 (2011)
  The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair—and optimal repair via both pathways. Our data and previous data suggest a two-stage model for! chromatin decondensation that facilitates DSB repair.
• Stalled Fork Rescue via Dormant Replication Origins in Unchallenged S Phase Promotes Proper Chromosome Segregation and Tumor Suppression
  - mol cell 41(5):543-553 (2011)
  Eukaryotic cells license far more origins than are actually used for DNA replication, thereby generating a large number of dormant origins. Accumulating evidence suggests that such origins play a role in chromosome stability and tumor suppression, though the underlying mechanism is largely unknown. Here, we show that a loss of dormant origins results in an increased number of stalled replication forks, even in unchallenged S phase in primary mouse fibroblasts derived from embryos homozygous for the Mcm4Chaos3 allele. We found that this allele reduces the stability of the MCM2-7 complex, but confers normal helicase activity in vitro. Despite the activation of multiple fork recovery pathways, replication intermediates in these cells persist into M phase, increasing the number of abnormal anaphase cells with lagging chromosomes and/or acentric fragments. These findings suggest that dormant origins constitute a major pathway for stalled fork recovery, contributing to faith! ful chromosome segregation and tumor suppression.
• Methionine Adenosyltransferase II Serves as a Transcriptional Corepressor of Maf Oncoprotein
  - mol cell 41(5):554-566 (2011)
  Protein methylation pathways comprise methionine adenosyltransferase (MAT), which produces S-adenosylmethionine (SAM) and SAM-dependent substrate-specific methyltransferases. However, the function of MAT in the nucleus is largely unknown. MafK represses or activates expression of heme oxygenase-1 (HO-1) gene, depending on its heterodimer partners. Proteomics analysis of MafK revealed its interaction with MATIIα, a MAT isozyme. MATIIα was localized in nuclei and found to form a dense network with chromatin-related proteins including Swi/Snf and NuRD complexes. MATIIα was recruited to Maf recognition element (MARE) at HO-1 gene. When MATIIα was knocked down in murine hepatoma cell line, expression of HO-1 was derepressed at both basal and induced levels. The catalytic activity of MATIIα, as well as its interacting factors such as MATIIβ, BAF53a, CHD4, and PARP1, was required for HO-1 repression. MATII serves as a transcriptional corepressor of MafK by interacting w! ith chromatin regulators and supplying SAM for methyltransferases.
• Structure of Lipid Kinase p110β/p85β Elucidates an Unusual SH2-Domain-Mediated Inhibitory Mechanism
  - mol cell 41(5):567-578 (2011)
  Phosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110β/p85β complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110β catalytic subunit, which is essential for lipid kinase activity. In vitro, p110β basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110β. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RT! K signaling specificities.
• U1 snRNA Directly Interacts with Polypyrimidine Tract-Binding Protein during Splicing Repression
  - mol cell 41(5):579-588 (2011)
  Splicing of the c-src N1 exon is repressed by the polypyrimidine tract-binding protein (PTB or PTBP1). During exon repression, the U1 snRNP binds properly to the N1 exon 5′ splice site but is made inactive by the presence of PTB. Examining the patterns of nuclease protection at this 5′ splice site, we find that the interaction of U1 is altered by the adjacent PTB. Interestingly, UV crosslinking identifies a direct contact between the pre-mRNA-bound PTB and the U1 snRNA. EMSA, ITC, and NMR studies show that PTB RRMs 1 and 2 bind the pyrimidine-rich internal loop of U1 snRNA stem loop 4. The PTB/U1 interaction prevents further assembly of the U1 snRNP with spliceosomal components downstream. This precise interaction between a splicing regulator and an snRNA component of the spliceosome points to a range of different mechanisms for splicing regulation.
• Dicer's Helicase Domain Discriminates dsRNA Termini to Promote an Altered Reaction Mode
  - mol cell 41(5):589-599 (2011)
  The role of Dicer's helicase domain is enigmatic, but in vivo it is required for processing certain endogenous siRNA, but not miRNA. By using Caenorhabditis elegans extracts or purified Drosophila Dicer-2 we compared activities of wild-type enzymes and those containing mutations in the helicase domain. We found the helicase domain was essential for cleaving dsRNA with blunt or 5′-overhanging termini, but not those with 3′ overhangs, as found on miRNA precursors. Further, blunt termini, but not 3′ overhangs, led to increased siRNAs from internal regions of dsRNA; this activity required ATP and a functional helicase domain. Our data suggest that blunt or 5′-overhanging termini engage Dicer's helicase domain to facilitate accumulation of siRNAs from internal regions of a dsRNA, an activity suited for processing long siRNA precursors of low abundance, but not necessary for the single cleavage required for miRNA processing.
• Coupled 5′ Nucleotide Recognition and Processivity in Xrn1-Mediated mRNA Decay
  - mol cell 41(5):600-608 (2011)
  Messenger RNA decay plays a central role in the regulation and surveillance of eukaryotic gene expression. The conserved multidomain exoribonuclease Xrn1 targets cytoplasmic RNA substrates marked by a 5′ monophosphate for processive 5′-to-3′ degradation by an unknown mechanism. Here, we report the crystal structure of an Xrn1-substrate complex. The single-stranded substrate is held in place by stacking of the 5′-terminal trinucleotide between aromatic side chains while a highly basic pocket specifically recognizes the 5′ phosphate. Mutations of residues involved in binding the 5′-terminal nucleotide impair Xrn1 processivity. The substrate recognition mechanism allows Xrn1 to couple processive hydrolysis to duplex melting in RNA substrates with sufficiently long single-stranded 5′ overhangs. The Xrn1-substrate complex structure thus rationalizes the exclusive specificity of Xrn1 for 5′-monophosphorylated substrates, ensuring fidelity of mRNA turnover, an! d posits a model for translocation-coupled unwinding of structured RNA substrates.
• USP47 Is a Deubiquitylating Enzyme that Regulates Base Excision Repair by Controlling Steady-State Levels of DNA Polymerase β
  - mol cell 41(5):609-615 (2011)
  DNA base excision repair (BER) is an essential cellular process required for genome stability, and misregulation of BER is linked to premature aging, increased rate of mutagenesis, and cancer. We have now identified the cytoplasmic ubiquitin-specific protease USP47 as the major enzyme involved in deubiquitylation of the key BER DNA polymerase (Pol β) and demonstrate that USP47 is required for stability of newly synthesized cytoplasmic Pol β that is used as a source for nuclear Pol β involved in DNA repair. We further show that knockdown of USP47 causes an increased level of ubiquitylated Pol β, decreased levels of Pol β, and a subsequent deficiency in BER, leading to accumulation of DNA strand breaks and decreased cell viability in response to DNA damage. Taken together, these data demonstrate an important role for USP47 in regulating DNA repair and maintaining genome integrity.