White Rose University Consortium logo
University of Leeds logo University of Sheffield logo York University logo

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

Johnson, R., Borde, V., Neale, M.J., Bishop-Bailey, A., North, M., Harris, S., Nicolas, A. and Goldman, A.S.H. (2007) Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair. PLoS Genetics, 3 (11). e223. ISSN 1553-7390

Full text available as:

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 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 dmc1Δ 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 dmc1Δ 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.

Item Type: Article
Copyright, Publisher and Additional Information: © 2007 Johnson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Institution: The University of Sheffield
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > School of Biological Sciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield)
Depositing User: Sheffield Import
Date Deposited: 27 Oct 2009 16:45
Last Modified: 15 Sep 2014 01:17
Published Version: http://dx.doi.org/10.1371/journal.pgen.0030223
Status: Published
Publisher: Public Library of Science
Refereed: Yes
Identification Number: doi: 10.1371/journal.pgen.0030223
URI: http://eprints.whiterose.ac.uk/id/eprint/10035

Actions (repository staff only: login required)