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J Biol Chem. 2017 Jul 14;292(28):11702-11713. doi: 10.1074/jbc.M117.794545. Epub 2017 May 27.

Human RAD52 interactions with replication protein A and the RAD51 presynaptic complex.

Author information

1
From the Department of Biochemistry & Molecular Biophysics, Columbia University, New York, New York 10032 and.
2
the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520.
3
From the Department of Biochemistry & Molecular Biophysics, Columbia University, New York, New York 10032 and ecg2108@cumc.columbia.edu.

Abstract

Rad52 is a highly conserved protein involved in the repair of DNA damage. Human RAD52 has been shown to mediate single-stranded DNA (ssDNA) and is synthetic lethal with mutations in other key recombination proteins. For this study, we used single-molecule imaging and ssDNA curtains to examine the binding interactions of human RAD52 with replication protein A (RPA)-coated ssDNA, and we monitored the fate of RAD52 during assembly of the presynaptic complex. We show that RAD52 binds tightly to the RPA-ssDNA complex and imparts an inhibitory effect on RPA turnover. We also found that during presynaptic complex assembly, most of the RPA and RAD52 was displaced from the ssDNA, but some RAD52-RPA-ssDNA complexes persisted as interspersed clusters surrounded by RAD51 filaments. Once assembled, the presence of RAD51 restricted formation of new RAD52-binding events, but additional RAD52 could bind once RAD51 dissociated from the ssDNA. Together, these results provide new insights into the behavior and dynamics of human RAD52 during presynaptic complex assembly and disassembly.

KEYWORDS:

DNA recombination; DNA repair; DNA-binding protein; microscopic imaging; single-molecule biophysics

PMID:
28551686
PMCID:
PMC5512066
DOI:
10.1074/jbc.M117.794545
[Indexed for MEDLINE]
Free PMC Article

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