Abstract
Homologous recombination is a conserved pathway for repairing double-stranded breaks, which are processed to yield single-stranded DNA overhangs that serve as platforms for presynaptic-complex assembly. Here we use single-molecule imaging to reveal the interplay between Saccharomyces cerevisiae RPA, Rad52 and Rad51 during presynaptic-complex assembly. We show that Rad52 binds RPA-ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 binding extends the ssDNA, and Rad52-RPA clusters remain interspersed along the presynaptic complex. These clusters promote additional binding of RPA and Rad52. Our work illustrates the spatial and temporal progression of the association of RPA and Rad52 with the presynaptic complex and reveals a new RPA-Rad52-Rad51-ssDNA intermediate, with implications for how the activities of Rad52 and RPA are coordinated with Rad51 during the later stages of recombination.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Binding Sites
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DNA Breaks, Double-Stranded
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DNA, Single-Stranded / metabolism*
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Homologous Recombination / genetics
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Protein Binding / genetics
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Rad51 Recombinase / genetics
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Rad51 Recombinase / metabolism*
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Rad52 DNA Repair and Recombination Protein / genetics
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Rad52 DNA Repair and Recombination Protein / metabolism*
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Recombinational DNA Repair / genetics*
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Replication Protein A / genetics
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Replication Protein A / metabolism*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
Substances
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DNA, Single-Stranded
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RAD52 protein, S cerevisiae
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RFA1 protein, S cerevisiae
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Rad52 DNA Repair and Recombination Protein
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Replication Protein A
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Saccharomyces cerevisiae Proteins
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RAD51 protein, S cerevisiae
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Rad51 Recombinase