Journal article
Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52–ssDNA complexes
Nucleic acids research, Vol.38(9), pp.2917-2930
05/2010
DOI: 10.1093/nar/gkp1249
PMCID: PMC2875008
PMID: 20081207
Abstract
Rad52 promotes the annealing of complementary strands of DNA bound by replication protein A (RPA) during discrete repair pathways. Here, we used a fluorescence resonance energy transfer (FRET) between two fluorescent dyes incorporated into DNA substrates to probe the mechanism by which human Rad52 (hRad52) interacts with and mediates annealing of ssDNA–hRPA complexes. Human Rad52 bound ssDNA or ssDNA–hRPA complex in two, concentration-dependent modes. At low hRad52 concentrations, ssDNA was wrapped around the circumference of the protein ring, while at higher protein concentrations, ssDNA was stretched between multiple hRad52 rings. Annealing by hRad52 occurred most efficiently when each complementary DNA strand or each ssDNA–hRPA complex was bound by hRad52 in a wrapped configuration, suggesting homology search and annealing occur via two hRad52–ssDNA complexes. In contrast to the wild type protein, hRad52
RQK/AAA
and hRad52
1–212
mutants with impaired ability to bind hRPA protein competed with hRPA for binding to ssDNA and failed to counteract hRPA-mediated duplex destabilization highlighting the importance of hRad52-hRPA interactions in promoting efficient DNA annealing.
Details
- Title: Subtitle
- Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52–ssDNA complexes
- Creators
- Jill M Grimme - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801Masayoshi Honda - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801Rebecca Wright - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801Yusuke Okuno - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801Eli Rothenberg - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801Alexander V Mazin - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801Taekjip Ha - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801Maria Spies - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801
- Resource Type
- Journal article
- Publication Details
- Nucleic acids research, Vol.38(9), pp.2917-2930
- Publisher
- Oxford University Press
- DOI
- 10.1093/nar/gkp1249
- PMID
- 20081207
- PMCID
- PMC2875008
- ISSN
- 0305-1048
- eISSN
- 1362-4962
- Language
- English
- Date published
- 05/2010
- Academic Unit
- Radiation Oncology; Biochemistry and Molecular Biology
- Record Identifier
- 9984024558402771
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