Hydrogen–deuterium exchange reveals a dynamic DNA-binding map of replication protein A. Issue 3 (14th January 2021)

• Record Type: Journal Article
• Authors: Ahmad, Faiz; Patterson, Angela; Deveryshetty, Jaigeeth; Mattice, Jenna R; Pokhrel, Nilisha; Bothner, Brian; Antony, Edwin
• Is Part Of: Nucleic acids research. Volume 49:Issue 3(2021)
• Page Start: 1455
• Page End: 1469
• Publication Date: 2021-01-14
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkaa1288
• Languages: English
• ISSNs: 0305-1048

Abstract

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. RPA binds ssDNA utilizing multiple oligosaccharide/oligonucleotide binding domains and based on their individual DNA binding affinities 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 ssDNA-driven dynamics of the individual domains of human RPA. As expected, ssDNA binding shows HDX changes in DBDs A, B, C, D and E. 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. Slower allosteric changes transpire in the protein–protein interaction domains and linker regions, and thus do not directly interact with ssDNA. Within a dynamics-based model for RPA, we propose that DBD-A and -B act as the dynamic half and DBD-C, -D and -E function as the less-dynamic half. Thus, segments of ssDNA buried under the dynamic half are likely more readily accessible to RPA-interacting proteins. Graphical Abstract: