Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, 63104, USA.
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, 59717, USA.
Nat Commun. 2022 Sep 2;13(1):5152. doi: 10.1038/s41467-022-32860-6.
Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.
复制蛋白 A（RPA）是一种异源三聚体复合物，可与单链 DNA（ssDNA）结合，并招募三十多种 RPA 相互作用蛋白，以协调 DNA 代谢的多个方面，包括 DNA 复制、修复和重组。Rtt105 是一种分子伴侣，可调节 RPA 的核定位。在这里，我们表明 Rtt105 结合到 RPA 的多个 DNA 结合和蛋白相互作用结构域，并使复合物构象稳定。在没有 ssDNA 的情况下，Rtt105 抑制 RPA 与 Rad52 的结合，从而防止与 RPA 相互作用蛋白的错误结合。当 ssDNA 存在时，Rtt105 促进高密度 RPA 核蛋白丝的形成，并在此过程中解离。游离的 Rtt105 通过抑制 RPA 的促进交换活性进一步稳定 RPA-ssDNA 丝。总的来说，我们的数据表明，Rtt105 将游离的 RPA 隔离在核内，以防止其与 RPA 相互作用蛋白过早结合，同时稳定 DNA 损伤部位的 RPA-ssDNA 丝。
