The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. ...Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3′-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.
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•RECQ5 binds to and stimulates MUS81-EME1 endonuclease•RECQ5 promotes mitotic DNA synthesis (MiDAS) at CFSs•RECQ5 removes RAD51 filaments protecting stalled replication forks at CFSs•Expression of CFSs depends on RECQ5 phosphorylation by CDK1
MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites in early mitosis to trigger DNA-repair synthesis, ensuring faithful chromosome segregation. Di Marco et al. show that RECQ5 eliminates RAD51 filaments from these structures to facilitate the access of MUS81-EME1 to DNA.
Unscheduled homologous recombination (HR) can lead to genomic instability, which greatly increases the threat of neoplastic transformation in humans. The F-box DNA helicase 1 (FBH1) is a 3'-5' DNA ...helicase with a putative function as a negative regulator of HR. It is the only known DNA helicase to contain an F-box, suggesting that one of its functions is to act as a ubiquitin ligase as part of an SCF (SKP1, CUL1 and F-box) complex. Here we report that the central player in HR, RAD51, is ubiquitylated by the SCF(FBH1) complex. Expression of an ubiquitylation-resistant form of RAD51 in human cells leads to hyperrecombination, as well as several phenotypes indicative of an altered response to DNA replication stress. These effects are likely to be mediated by the enhanced nuclear matrix association of the ubiquitylation-resistant RAD51. These data are consistent with FBH1 acting as a negative regulator of RAD51 function in human cells.
Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence ...of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.
Bloom's syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. We describe crystal structures of ...the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. We show an unexpected nucleotide-dependent interaction of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM-DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates.
Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ ...helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathological features of Bloom syndrome, we analyzed mRNA and microRNA (miRNA) expression in fibroblasts from individuals with Bloom syndrome and in BLM-depleted control fibroblasts. We identified mRNA and miRNA expression differences in Bloom syndrome patient and BLM-depleted cells. Differentially expressed mRNAs are connected with cell proliferation, survival, and molecular mechanisms of cancer, and differentially expressed miRNAs target genes involved in cancer and in immune function. These and additional altered functions or pathways may contribute to the proportional dwarfism, elevated cancer risk, immune dysfunction, and other features observed in Bloom syndrome individuals. BLM binds to G-quadruplex (G4) DNA, and G4 motifs were enriched at transcription start sites (TSS) and especially within first introns (false discovery rate ≤ 0.001) of differentially expressed mRNAs in Bloom syndrome compared with normal cells, suggesting that G-quadruplex structures formed at these motifs are physiologic targets for BLM. These results identify a network of mRNAs and miRNAs that may drive the pathogenesis of Bloom syndrome.
The ATR kinase is a master regulator of the cellular response to DNA replication stress. Activation of ATR relies on dual pathways involving the TopBP1 and ETAA1 proteins, both of which harbor ...ATR-activating domains (AADs). However, the exact contribution of the recently discovered ETAA1 pathway to ATR signaling in different contexts remains poorly understood. Here, using an unbiased CRISPR-Cas9-based genome-scale screen, we show that the ATR-stimulating function of ETAA1 becomes indispensable for cell fitness and chromosome stability when the fidelity of DNA replication is compromised. We demonstrate that the ATR-activating potential of ETAA1 is controlled by cell cycle- and replication stress-dependent phosphorylation of highly conserved residues within its AAD, and that the stimulatory impact of these modifications is required for the ability of ETAA1 to prevent mitotic chromosome abnormalities following replicative stress. Our findings suggest an important role of ETAA1 in protecting against genome instability arising from incompletely duplicated DNA via regulatory control of its ATR-stimulating potential.
Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high ...specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.
Fanconi anemia (FA) is a heritable human cancer-susceptibility disorder, delineating a genetically heterogenous pathway for the repair of replication-blocking lesions such as interstrand DNA ...cross-links. Here we demonstrate that one component of this pathway, FANCJ, is a structure-specific DNA helicase that dissociates guanine quadruplex DNA (G4 DNA) in vitro. Moreover, in contrast with previously identified G4 DNA helicases, such as the Bloom's helicase (BLM), FANCJ unwinds G4 substrates with 5′–3′ polarity. In the FA-J human patient cell line EUFA0030 the loss of FANCJ G4 unwinding function correlates with the accumulation of large genomic deletions in the vicinity of sequences, which match the G4 DNA signature. Together these findings support a role for FANCJ in the maintenance of potentially unstable genomic G/C tracts during replication.
Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a ...repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.
Background: Homologous recombination is regulated both positively and negatively in eukaryotic cells to suppress genomic instability.
Results: FBH1 can disrupt RAD51 filaments in vitro and suppresses formation of spontaneous RAD51 foci in mammalian cells. In cells defective for FBH1, hyper-recombination is observed.
Conclusion: FBH1 is a negative regulator of homologous recombination.
Significance: RAD51 activity must be carefully controlled to preserve genomic integrity.
The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, we show that the Bloom syndrome gene product, BLM, counteracts ...CtIP/MRE11-dependent long-range deletions (>200 bp) generated by alternative end-joining (A-EJ). BLM represses A-EJ in an epistatic manner with 53BP1 and RIF1 and is required for ionizing-radiation-induced 53BP1 focus assembly. Conversely, in the absence of 53BP1 or RIF1, BLM promotes formation of A-EJ long deletions, consistent with a role for BLM in DSB end resection. These data highlight a dual role for BLM that influences the DSB repair pathway choice: (1) protection against CtIP/MRE11 long-range deletions associated with A-EJ and (2) promotion of DNA resection. These antagonist roles can be regulated, according to cell-cycle stage, by interacting partners such as 53BP1 and TopIII, to avoid unscheduled resection that might jeopardize genome integrity.
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•BLM protects against CtIP-dependent A-EJ•BLM protects against CtIP-dependent sister chromatid exchanges•BLM promotes the accumulation of 53BP1 at DSBs•53BP1 protects against both CtIP- and BLM- mediated A-EJ long-range deletions
The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, Lopez and colleagues show a dual role for the Bloom syndrome gene product, BLM, that influences the DSB repair pathway choice in two ways: (1) protection against long-range deletions associated with DSB end-joining and (2) promotion of DNA resection. These antagonistic roles can be regulated, according to cell-cycle stage, by interacting partners, to avoid unscheduled resection that might jeopardize genome integrity.
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