Partial wrapping of single-stranded DNA by replication protein A and modulation through phosphorylation

Rahul Chadda, Vikas Kaushik, Iram Munir Ahmad, Jaigeeth Deveryshetty, Alex S. Holehouse, Snorri Th Sigurdsson, Gargi Biswas, Yaakov Levy, Brian Bothner, Richard B. Cooley, Ryan A. Mehl, Reza Dastvan, Sofia Origanti, Edwin Antony

Research output: Contribution to journalArticlepeer-review

Abstract

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA–ssDNA complexes using single-molecule FRET and double electron–electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Original languageEnglish
Pages (from-to)11626-11640
Number of pages15
JournalNucleic acids research
Volume52
Issue number19
DOIs
StatePublished - Oct 28 2024

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