TY - JOUR
T1 - Partial wrapping of single-stranded DNA by replication protein A and modulation through phosphorylation
AU - Chadda, Rahul
AU - Kaushik, Vikas
AU - Ahmad, Iram Munir
AU - Deveryshetty, Jaigeeth
AU - Holehouse, Alex S.
AU - Sigurdsson, Snorri Th
AU - Biswas, Gargi
AU - Levy, Yaakov
AU - Bothner, Brian
AU - Cooley, Richard B.
AU - Mehl, Ryan A.
AU - Dastvan, Reza
AU - Origanti, Sofia
AU - Antony, Edwin
N1 - Publisher Copyright:
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2024/10/28
Y1 - 2024/10/28
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85208128664&partnerID=8YFLogxK
U2 - 10.1093/nar/gkae584
DO - 10.1093/nar/gkae584
M3 - Article
C2 - 38989614
AN - SCOPUS:85208128664
SN - 0305-1048
VL - 52
SP - 11626
EP - 11640
JO - Nucleic acids research
JF - Nucleic acids research
IS - 19
ER -