Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Nucleic Acids Res. 2010 May;38(9):2917-30. doi: 10.1093/nar/gkp1249. Epub 2010 Jan 16.
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.
Rad52 促进复制蛋白 A（RPA）结合的 DNA 互补链在不同修复途径中的退火。在这里，我们使用荧光共振能量转移（FRET）在掺入 DNA 底物中的两个荧光染料之间进行探测，以研究人 Rad52（hRad52）与 ssDNA-hRPA 复合物相互作用并介导 ssDNA 退火的机制。人 Rad52 以两种浓度依赖性模式结合 ssDNA 或 ssDNA-hRPA 复合物。在低 hRad52 浓度下，ssDNA 围绕蛋白质环的周长缠绕，而在较高的蛋白质浓度下，ssDNA 在多个 hRad52 环之间伸展。当每个互补 DNA 链或每个 ssDNA-hRPA 复合物都以包裹的构型被 hRad52 结合时，hRad52 的退火效率最高，这表明同源搜索和退火通过两个 hRad52-ssDNA 复合物发生。与野生型蛋白相比，hRad52(RQK/AAA)和 hRad52(1-212)突变体结合 hRPA 蛋白的能力受损，与 hRPA 竞争结合 ssDNA，并且不能抵消 hRPA 介导的双链体不稳定，突出了 hRad52-hRPA 相互作用在促进有效 DNA 退火中的重要性。
