Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair
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 requir...
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| Format: | Online |
| Language: | English |
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Public Library of Science
2007
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2098809/ |
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pubmed-2098809 |
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pubmed-20988092007-11-29 Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair Johnson, Rebecca Borde, Valérie Neale, Matthew J Bishop-Bailey, Anna North, Matthew Harris, Sheila Nicolas, Alain Goldman, Alastair S. H Research Article 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. Public Library of Science 2007-11 2007-11-30 /pmc/articles/PMC2098809/ /pubmed/18081428 http://dx.doi.org/10.1371/journal.pgen.0030223 Text en © 2007 Johnson et al. http://creativecommons.org/licenses/by/4.0/ 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 properly credited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Johnson, Rebecca Borde, Valérie Neale, Matthew J Bishop-Bailey, Anna North, Matthew Harris, Sheila Nicolas, Alain Goldman, Alastair S. H |
| spellingShingle |
Johnson, Rebecca Borde, Valérie Neale, Matthew J Bishop-Bailey, Anna North, Matthew Harris, Sheila Nicolas, Alain Goldman, Alastair S. H Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair |
| author_facet |
Johnson, Rebecca Borde, Valérie Neale, Matthew J Bishop-Bailey, Anna North, Matthew Harris, Sheila Nicolas, Alain Goldman, Alastair S. H |
| author_sort |
Johnson, Rebecca |
| title |
Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair |
| title_short |
Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair |
| title_full |
Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair |
| title_fullStr |
Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair |
| title_full_unstemmed |
Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair |
| title_sort |
excess single-stranded dna inhibits meiotic double-strand break repair |
| description |
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. |
| publisher |
Public Library of Science |
| publishDate |
2007 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2098809/ |
| _version_ |
1611408485618024448 |