Browsing by Author "Deveryshetty, Jaigeeth"

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Hydrogen–deuterium exchange reveals a dynamic DNA-binding map of replication protein A
(Oxford University Press, 2021-01) Ahmad, Faiz; Patterson, Angela; Deveryshetty, Jaigeeth; Mattice, Jenna R; Pokhrel, Nilisha; Bothner, Brian; Antony, Edwin
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.
Rtt105 regulates RPA function by configurationally stapling the flexible domains
(Springer Science and Business Media LLC, 2022-09) Kuppa, Sahiti; Deveryshetty, Jaigeeth; Chadda, Rahul; Mattice, Jenna R.; Pokhrel, Nilisha; Kaushik, Vikas; Patterson, Angela; Dhingra, Nalini; Pangeni, Sushil; Sadauskas, Marisa K.; Shiekh, Sajad; Balci, Hamza; Ha, Taekjip; Zhao, Xiaolan; Bothner, Brian; Antony, Edwin
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.
Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes
(Springer Science and Business Media LLC, 2023-10) Deveryshetty, Jaigeeth; Chadda, Rahul; Mattice, Jenna R.; Karunakaran, Simrithaa; Rau, Michael J.; Basore, Katherine; Pokhrel, Nilisha; Englander, Noah; Fitzpatrick, James A. J.; Bothner, Brian; Antony, Edwin
Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.