Mattice, J. | bioRxiv Science

bioRxiv2022

Rtt105 configurationally staples RPA and blocks facilitated exchange and interactions with RPA-interacting proteins

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins (RIPs) to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Whether and how Rtt105 regulates the activities of RPA is poorly understood. 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 (RIPs). 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 RPA facilitated exchange. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely RIP interaction, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

biochemistry↗

bioRxiv2020

Hydrogen-deuterium exchange reveals a dynamic DNA binding map of Replication Protein A

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes including DNA replication, repair, and recombination. RPA binds ssDNA utilizing six oligosaccharide/oligonucleotide binding (OB) domains within a heterotrimeric complex of RPA70, RPA32 and RPA14 subunits. Based on their DNA binding affinities they are classified as high versus low-affinity DNA binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS) we assessed the contacts and dynamics of the individual domains of human RPA to determine the landscape of conformational changes upon binding to ssDNA. As expected, ssDNA interacts with the major DBDs (A, B, C, and D). However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. DNA-dependent HDX kinetics are also captured for DBD-D and DBD-E. Slower allosteric changes transpire in the protein-protein interaction domains and the linker regions. We propose a dynamics-based DNA binding model for RPA utilizing a dynamic half and a less-dynamic half.

biochemistry↗