Mechanism of replication fork reversal and protection by human RAD51 and RAD51 paralogs
TL;DR: It is shown that the protective function of RAD51 unexpectedly depends on its binding to double-stranded DNA, and higher RAD51 concentrations are required for DNA protection compared to reversal, and the mechanisms of the non-canonical functions of RAD 51 and paralogs in replication fork reversal and protection are defined.
Abstract: RAD51 functions in DNA double-strand break repair by homologous recombination, and by a yet undefined mechanism in the metabolism of challenged replication forks. Here we show that RAD51 directly and specifically promotes the strand annealing and branch migration activities of SMARCAL1 and ZRANB3 but not HLTF, stimulating thus fork reversal. We also find that the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), additionally stimulates SMARCAL1 and ZRANB3 in fork remodeling. DNA binding by RAD51 is required, and the interplay of RAD51, paralogs and the fork remodelers involves direct physical interactions. Upon reversal, RAD51 protects replication forks from degradation by MRE11, DNA2 and EXO1 nucleases. We show that the protective function of RAD51 unexpectedly depends on its binding to double-stranded DNA, and higher RAD51 concentrations are required for DNA protection compared to reversal. Together, we define the mechanisms of the non-canonical functions of RAD51 and paralogs in replication fork reversal and protection.
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TL;DR: Last week the BMJ and Nuffield Trust brought together some of the leading voices in healthcare to consider what life in the NHS will be like after the Health and Social Care Bill passes into legislation.
Abstract: Last week the BMJ and Nuffield Trust brought together some of the leading voices in healthcare to consider what life in the NHS will be like after the Health and Social Care Bill passes into legislation. Rebecca Coombes presents the highlights
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05 Jul 2018
TL;DR: The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2dependent histone chaperone activity as mentioned in this paper.
Abstract: Summary Components of the Fanconi anemia and homologous recombination pathways play a vital role in protecting newly replicated DNA from uncontrolled nucleolytic degradation, safeguarding genome stability. Here we report that histone methylation by the lysine methyltransferase SETD1A is crucial for protecting stalled replication forks from deleterious resection. Depletion of SETD1A sensitizes cells to replication stress and leads to uncontrolled DNA2-dependent resection of damaged replication forks. The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2-dependent histone chaperone activity. Suppressing H3K4 methylation or expression of a chaperone-defective FANCD2 mutant leads to loss of RAD51 nucleofilament stability and severe nucleolytic degradation of replication forks. Our work identifies epigenetic modification and histone mobility as critical regulatory mechanisms in maintaining genome stability by restraining nucleases from irreparably damaging stalled replication forks.
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TL;DR: In this paper, the kinase ATR (ATM- and Rad3-related) stabilizes and helps to restart stalled replication forks, avoiding the generation of DNA damage and genome instability.
Abstract: Replication stress is a complex phenomenon that has serious implications for genome stability, cell survival and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATR (ATM- and Rad3-related). Along with its downstream effectors, ATR stabilizes and helps to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding this response may be key to diagnosing and treating human diseases caused by defective responses to replication stress.
1,492 citations
TL;DR: Using single-molecule DNA fiber analysis, it is shown that nascent replication tracts created before fork stalling with hydroxyurea are degraded in the absence of BRCA2 but are stable in wild-type cells.
1,001 citations
TL;DR: It is speculated that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.
Abstract: Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.
827 citations
TL;DR: A repair-independent requirement for FA genes, including FANCD2, and BRCA1 in protecting stalled replication forks from degradation is shown, implying a unified molecular mechanism for repair- independent functions of FA, RAD51, and PSA1/2 proteins in preventing genomic instability and suppressing tumorigenesis.
755 citations
TL;DR: The BRC repeat mimics a motif in RAD51 that serves as an interface for oligomerization between individual RAD51 monomers, thus enabling BRCA2 to control the assembly of the RAD51 nucleoprotein filament, which is essential for strand-pairing reactions during DNA recombination.
Abstract: The breast cancer susceptibility protein BRCA2 controls the function of RAD51, a recombinase enzyme, in pathways for DNA repair by homologous recombination. We report here the structure of a complex between an evolutionarily conserved sequence in BRCA2 (the BRC repeat) and the RecA-homology domain of RAD51. The BRC repeat mimics a motif in RAD51 that serves as an interface for oligomerization between individual RAD51 monomers, thus enabling BRCA2 to control the assembly of the RAD51 nucleoprotein filament, which is essential for strand-pairing reactions during DNA recombination. The RAD51 oligomerization motif is highly conserved among RecA-like recombinases, highlighting a common evolutionary origin for the mechanism of nucleoprotein filament formation, mirrored in the BRC repeat. Cancer-associated mutations that affect the BRC repeat disrupt its predicted interaction with RAD51, yielding structural insight into mechanisms for cancer susceptibility.
665 citations