Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52-ssDNA complexes.
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.
Grimme
Jill M
JM
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Honda
Masayoshi
M
Wright
Rebecca
R
Okuno
Yusuke
Y
Rothenberg
Eli
E
Mazin
Alexander V
AV
Ha
Taekjip
T
Spies
Maria
M
D001667:Binding, Competitive; D004277:DNA, Single-Stranded / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D051721:Rad52 DNA Repair and Recombination Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051716:Replication Protein A / Q000378:metabolism
D004277:DNA, Single-Stranded; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A