Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair

Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom.
PLoS Genetics (Impact Factor: 7.53). 12/2007; 3(11):e223. DOI: 10.1371/journal.pgen.0030223
Source: PubMed


During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

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    • "Finally, our genetic reporter assay demonstrated an increase in intersister DSB repair at one locus. Our data and those of other groups are consistent with recombination checkpoint signaling being sensitive to unrepaired DSB levels, such that more DSBs trigger checkpoint arrest in a greater proportion of the cell population (Malkova et al. 1996; Bhalla and Dernburg 2005; MacQueen et al. 2005; Johnson et al. 2007; Callender and Hollingsworth 2010; Goldfarb and Lichten 2010). In wild-type cells, DSBs are quickly repaired and the Mek1-mediated checkpoint delay is transient. "
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    ABSTRACT: In most organisms, the segregation of chromosomes during the first meiotic division is dependent upon at least one crossover (CO) between each pair of homologous chromosomes. COs can result from chromosome double-strand breaks (DSBs) that are induced and preferentially repaired using the homologous chromosome as a template. The PCH2 gene of budding yeast is required to establish proper meiotic chromosome axis structure and to regulate meiotic interhomolog DSB repair outcomes. These roles appear conserved in the mouse ortholog of PCH2, Trip13, which is also involved in meiotic chromosome axis organization and the regulation of DSB repair. Using a combination of genetic and physical assays to monitor meiotic DSB repair, we present data consistent with pch2Δ mutants showing defects in suppressing intersister DSB repair. These defects appear most pronounced in dmc1Δ mutants, which are defective for interhomolog repair, and explain the previously reported observation that pch2Δ dmc1Δ cells can complete meiosis. Results from genetic epistasis analyses involving spo13Δ, rad54Δ, and mek1/MEK1 alleles and an intersister recombination reporter assay are also consistent with Pch2 acting to limit intersister repair. We propose a model in which Pch2 is required to promote full Mek1 activity and thereby promotes interhomolog repair.
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    • "Extensive resection during meiosis could be deleterious because if all DSBs were resected for ≥3kb on both sides a typical meiotic cell would expose ~1.5 Mb of ssDNA; this will likely include repetitive DNA elements that could undergo single-strand annealing (leading to deletions), or engage in non-allelic recombination that may cause rearrangements and aberrant chromosome segregation. Furthermore, excess ssDNA may have an inhibitory effect on normal DSB-repair during meiosis (Johnson et al., 2007). "
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    • "The arg4-vde allele receives DSBs independently of MEK1 status. (A) The chromosome V ura3::arg4-vde reporter cassette containing the VDE–DSB1 and the chromosome VIII region containing arg4-nsp,bgl site as described previously (22,23). Following restriction endonuclease digestion with EcoRV (RV) and BglII and probing a Southern blot with probe (Pr), (B) the chromosome V arg4-vde parental fragment (P) and chromosome VIII arg4-nsp,bgl fragment (LC) are isolated from other species (not shown). "
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