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Nat Struct Mol Biol. 2014 Oct;21(10):893-900. doi: 10.1038/nsmb.2886. Epub 2014 Sep 7.

Protein dynamics during presynaptic-complex assembly on individual single-stranded DNA molecules.

Author information

1
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA.
2
Department of Biological Sciences, Columbia University, New York, New York, USA.
3
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA.
4
1] Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA. [2] Howard Hughes Medical Institute, Columbia University, New York, New York, USA.

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.

PMID:
25195049
PMCID:
PMC4190069
DOI:
10.1038/nsmb.2886
[Indexed for MEDLINE]
Free PMC Article

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