DMCA
Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair
| Citations: | 4 - 1 self |
BibTeX
@MISC{[¤a_excesssingle-stranded,
author = {Rebecca Johnson [¤a and Valérie Borde and Matthew J. Neale [¤b and Anna Bishop-bailey and Matthew North ¤c and Sheila Harris ¤d and Alain Nicolas and Alastair S. H. Goldman},
title = {Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair},
year = {}
}
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Abstract
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 Dmc1dependent 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 dmc1D 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 dmc1D 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,
Keyphrases
dmc1d cell vma1-derived endonuclease single-strand annealing vde-dsb repair spo11-dsb formation single-strand binding complex replication protein crossover formation homologous recombination dsb repair homologous chromosome unrepaired single-stranded dna long tract resection chromatin immunoprecipitation experiment self-inflicted dna double-strand break direct repeat sequence site-specific dsbs abolish resection different reporter cassette reca orthologue large quantity first meiotic division dsbs sequester resection machinery protein spo11 dmc1dependent dna strand invasion step repeat sequence dmc1 strand-exchange protein repair block gene conversion ssdna binding protein
