Break-induced replication (BIR) is the pathway of homologous recombination (HR) conserved from phages to eukaryotes that serves to repair DNA breaks that have only one end. BIR contributes to the ...repair of broken replication forks and allows telomere lengthening in the absence of telomerase. Nonallelic BIR may lead to translocations and other chromosomal rearrangements. In addition, BIR initiated at sites of microhomology can generate copy number variations (CNVs) and complex chromosomal changes. The level of mutagenesis associated with DNA synthesis in BIR is significantly higher than during normal replication. These features make BIR a likely pathway to promote bursts of genetic changes that fuel cancer progression and evolution.
The breast cancer susceptibility proteins BRCA1 and BRCA2 have emerged as key stabilizing factors for the maintenance of replication fork integrity following replication stress. In their absence, ...stalled replication forks are extensively degraded by the MRE11 nuclease, leading to chemotherapeutic sensitivity. Here we report that BRCA proteins prevent nucleolytic degradation by protecting replication forks that have undergone fork reversal upon drug treatment. The unprotected regressed arms of reversed forks are the entry point for MRE11 in BRCA-deficient cells. The CtIP protein initiates MRE11-dependent degradation, which is extended by the EXO1 nuclease. Next, we show that the initial limited resection of the regressed arms establishes the substrate for MUS81 in BRCA2-deficient cells. In turn, MUS81 cleavage of regressed forks with a ssDNA tail promotes POLD3-dependent fork rescue. We propose that targeting this pathway may represent a new strategy to modulate BRCA2-deficient cancer cell response to chemotherapeutics that cause fork degradation.BRCA proteins have emerged as key stabilizing factors for the maintenance of replication forks following replication stress. Here the authors describe how reversed replication forks are degraded in the absence of BRCA2, and a MUS81 and POLD3-dependent mechanism of rescue following the withdrawal of genotoxic agent.
DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two‐ended DNA double‐strand breaks (DSBs) are usually repaired by gene conversion with a short patch ...of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single‐ended DSBs are repaired by break‐induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two‐ended DSBs. Here, we demonstrate that BIR is suppressed at two‐ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D‐loop unwinding helicase Mph1, and (iii) Mre11‐Rad50‐Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans.
Synopsis
While mutagenic break‐induced replication (BIR) repairs single‐ended DNA double‐strand breaks (DSBs), it needs to be suppressed at two‐ended breaks to ensure their faithful repair via gene conversion. Here, the coordinated usage of both DSB ends by ssDNA annealing and resection pathways is found to restrain unwanted BIR in budding yeast.
Rad52‐ and Rad59‐mediated ssDNA annealing suppresses BIR by promoting second‐end capture.
The Mre11‐Rad50‐Xrs2 complex suppresses BIR by promoting the synchronous formation of ssDNA at both ends of a break.
ssDNA annealing is less important for DSB repair and preventing BIR when homology is long.
Repair by BIR is not equally initiated at different genomic loci.
Coordinated usage of both DNA break ends via annealing proteins Rad52/59, D‐loop‐unwinding Mph1 helicase and the MRX complex ensures their repair by gene conversion, rather than by BIR operating at single‐ended breaks.
Single-stranded DNA (ssDNA) commonly occurs as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream ...factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors outcompete RPA to access ssDNA. Using the budding yeast Saccharomyces cerevisiae as a model system, we find that Dna2 - a key nuclease in DNA replication and repair - employs a bimodal interface to act with RPA both in cis and in trans. The cis-activity makes RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA delivers its bound ssDNA to Dna2. On the other hand, activity in trans is mediated by an acidic patch on Dna2, which enables it to function with a sub-optimal amount of RPA, or to overcome DNA secondary structures. The trans-activity mode is not required for cell viability, but is necessary for effective double strand break (DSB) repair.
Chromosome structural changes with nonrecurrent endpoints associated with genomic disorders offer windows into the mechanism of origin of copy number variation (CNV). A recent report of nonrecurrent ...duplications associated with Pelizaeus-Merzbacher disease identified three distinctive characteristics. First, the majority of events can be seen to be complex, showing discontinuous duplications mixed with deletions, inverted duplications, and triplications. Second, junctions at endpoints show microhomology of 2-5 base pairs (bp). Third, endpoints occur near pre-existing low copy repeats (LCRs). Using these observations and evidence from DNA repair in other organisms, we derive a model of microhomology-mediated break-induced replication (MMBIR) for the origin of CNV and, ultimately, of LCRs. We propose that breakage of replication forks in stressed cells that are deficient in homologous recombination induces an aberrant repair process with features of break-induced replication (BIR). Under these circumstances, single-strand 3' tails from broken replication forks will anneal with microhomology on any single-stranded DNA nearby, priming low-processivity polymerization with multiple template switches generating complex rearrangements, and eventual re-establishment of processive replication.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes ...remain unknown. We monitored 5′-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5′-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5′ degradation, whereas Sgs1 and Dna2 degrade 5′ strands exposing long 3′ strands. Deletion of
SGS1 or
DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In
exo1Δ
sgs1Δ double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.
Most spontaneous DNA double-strand breaks (DSBs) result from replication-fork breakage. Break-induced replication (BIR), a genome rearrangement–prone repair mechanism that requires the Pol32/POLD3 ...subunit of eukaryotic DNA Polδ, was proposed to repair broken forks, but how genome destabilization is avoided was unknown. We show that broken fork repair initially uses error-prone Pol32-dependent synthesis, but that mutagenic synthesis is limited to within a few kilobases from the break by Mus81 endonuclease and a converging fork. Mus81 suppresses template switches between both homologous sequences and diverged human Alu repetitive elements, highlighting its importance for stability of highly repetitive genomes. We propose that lack of a timely converging fork or Mus81 may propel genome instability observed in cancer.
Complex genomic rearrangements (CGRs) are a hallmark of many human diseases. Recently, CGRs were suggested to result from microhomology-mediated break-induced replication (MMBIR), a replicative ...mechanism involving template switching at positions of microhomology. Currently, the cause of MMBIR and the proteins mediating this process remain unknown. Here, we demonstrate in yeast that a collapse of homology-driven break-induced replication (BIR) caused by defective repair DNA synthesis in the absence of Pif1 helicase leads to template switches involving 0–6 nt of homology, followed by resolution of recombination intermediates into chromosomal rearrangements. Importantly, we show that these microhomology-mediated template switches, indicative of MMBIR, are driven by translesion synthesis (TLS) polymerases Polζ and Rev1. Thus, an interruption of BIR involving fully homologous chromosomes in yeast triggers a switch to MMBIR catalyzed by TLS polymerases. Overall, our study provides important mechanistic insights into the initiation of MMBIR associated with genomic rearrangements, similar to those promoting diseases in humans.
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•Collapse of DNA repair synthesis triggers a switch from BIR to MMBIR•Transition from BIR to MMBIR leads to chromosomal rearrangements•MMBIR is driven by translesion synthesis polymerases Polζ and Rev1•Template switches associated with MMBIR involve 0 to 6 nt of homology
Microhomology-mediated break-induced replication (MMBIR) is a poorly characterized molecular mechanism leading to the formation of complex chromosomal rearrangements associated with human diseases. Here, Sakofsky and colleagues demonstrate in yeast that MMBIR is initiated by the collapse of homology-mediated BIR and is driven by translesion synthesis polymerases Polζ and Rev1.
If not properly processed and repaired, DNA double-strand breaks (DSBs) can give rise to deleterious chromosome rearrangements, which could ultimately lead to the tumour phenotype. DSB ends are ...resected in a 5′ to 3′ fashion in cells, to yield single-stranded DNA (ssDNA) for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination. The resection process involves redundant pathways consisting of nucleases, DNA helicases and associated proteins. Being guided by recent genetic studies, we have reconstituted the first eukaryotic ATP-dependent DNA end-resection machinery comprising the Saccharomyces cerevisiae Mre11-Rad50-Xrs2 (MRX) complex, the Sgs1-Top3-Rmi1 complex, Dna2 protein and the heterotrimeric ssDNA-binding protein RPA. Here we show that DNA strand separation during end resection is mediated by the Sgs1 helicase function, in a manner that is enhanced by Top3-Rmi1 and MRX. In congruence with genetic observations, although the Dna2 nuclease activity is critical for resection, the Mre11 nuclease activity is dispensable. By examining the top3 Y356F allele and its encoded protein, we provide evidence that the topoisomerase activity of Top3, although critical for the suppression of crossover recombination, is not needed for resection either in cells or in the reconstituted system. Our results also unveil a multifaceted role of RPA, in the sequestration of ssDNA generated by DNA unwinding, enhancement of 5′ strand incision, and protection of the 3′ strand. Our reconstituted system should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) ...template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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