Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52ssDNA complexes

Author: Grimme Jill M.  

Publisher: Oxford University Press

ISSN: 1362-4962

Source: Nucleic Acids Research, Vol.38, Iss.9, 2010-05, pp. : 2917-2930

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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 ssDNAhRPA complexes. Human Rad52 bound ssDNA or ssDNAhRPA 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 ssDNAhRPA complex was bound by hRad52 in a wrapped configuration, suggesting homology search and annealing occur via two hRad52ssDNA complexes. In contrast to the wild type protein, hRad52RQK/AAA and hRad521212 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.