Nucleic Acids Research Advance Access published online on January 16, 2010
Nucleic Acids Research, doi:10.1093/nar/gkp1249
Genome Integrity, Repair and Replication |
Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52–ssDNA complexes
1Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, 2US Army Corps of Engineers, Construction Engineering Research Laboratory, Champaign, IL, 61822, 3Howard Hughes Medical Institute, Urbana, IL 61801, 4Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102-1192, 5Department of Physics and 6Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
*To whom correspondence should be addressed. Tel: +1 217 244 9493; Fax: +1 217 244 5858; Email: mspies{at}life.illinois.edu
Received July 31, 2009. Revised December 17, 2009. Accepted December 27, 2009.
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, hRad52RQK/AAA and hRad521–212 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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.