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

Rahul ChaddaVikas KaushikIram Munir AhmadJaigeeth DeveryshettyAlex S HolehouseSnorri Th SigurdssonGargi BiswasYaakov LevyBrian BothnerRichard B CooleyRyan A MehlReza DastvanSofia OrigantiEdwin Antony
Published in: Nucleic acids research (2024)
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.
Keyphrases
  • single molecule
  • dna binding
  • circulating tumor
  • binding protein
  • cell free
  • transcription factor
  • atomic force microscopy
  • nucleic acid
  • dna methylation
  • mass spectrometry
  • quantum dots
  • structural basis
  • high speed