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

Abstract

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.