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Rapid Long-distance Migration of RPA on Single Stranded DNA Occurs Through Intersegmental Transfer Utilizing

Sushil Pangeni1, Gargi Biswas2, Vikas Kaushik3

  • 1TC Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA; Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.

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Replication Protein A (RPA) is crucial for DNA metabolism. This study reveals RPA

Keywords:
DiffusionMD simulationReplication protein A (RPA)Single Molecule Biophysics

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Genetics

Background:

  • Replication Protein A (RPA) is a heterotrimeric protein essential for DNA replication, repair, and recombination.
  • RPA's flexibility, due to its six oligosaccharide/oligonucleotide (OB) domains and linkers, allows multiple configurations that modulate its function.

Purpose of the Study:

  • To investigate the diffusional migration of single RPA molecules on single-stranded DNA (ssDNA) under tension.
  • To understand how DNA tension and salt concentration affect RPA's ssDNA binding and migration dynamics.

Main Methods:

  • Single-molecule confocal fluorescence microscopy
  • Optical tweezers
  • Coarse-grained molecular dynamics simulations

Main Results:

  • RPA diffusion coefficient is highest at 3 pN tension and 100 mM KCl, decreasing with increased tension or salt concentration.
  • Tension hinders intersegmental transfer by DNA stretching; salt increases RPA-ssDNA binding site size and interaction energy.
  • Deletion of the RPA trimeric core increased RPA mobility 15-fold while retaining significant ssDNA binding.

Conclusions:

  • RPA-ssDNA interactions are remodeled by tension and salt concentration, influencing RPA's accessibility in DNA metabolic processes.
  • Intersegmental transfer events, involving transient bridging of distant DNA sites, were estimated in size and frequency.
  • RPA crowding effects on migration were characterized, providing insights into DNA repair and replication mechanisms.