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

• Published in: Journal of molecular biology Vol. 436; no. 6; p. 168491
• Main Authors: Pangeni, Sushil, Biswas, Gargi, Kaushik, Vikas, Kuppa, Sahiti, Yang, Olivia, Lin, Chang-Ting, Mishra, Garima, Levy, Yaakov, Antony, Edwin, Ha, Taekjip
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 15.03.2024
• Subjects: Diffusion; DNA Replication; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; energy; MD simulation; molecular biology; molecular dynamics; oligonucleotides; oligosaccharides; Protein Binding - genetics; Replication protein A (RPA); Replication Protein A - chemistry; Replication Protein A - genetics; Replication Protein A - metabolism; Single Molecule Biophysics; single-stranded DNA; MD simulation; Single Molecule Biophysics; Replication protein A (RPA); Diffusion
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2024.168491

[Display omitted] •Yeast replication protein A(yRPA) diffuses on the single stranded DNA through intersegmental transfer where two distant sites on the DNA are momentarily bridged by RPA through its multivalent interaction with DNA.•We further dissect the contributions of the trimerization core of RPA where deletion of RPA trimeric core made RPA 15-fold more mobile.•yRPA diffusion coefficient on ssDNA decreases dramatically with increasing DNA tension and decreases with increasing ionic strength as revealed by both experimental and computational approaches.•Coarse grained molecular dynamics simulations help to estimate the size of intersegmental transfer and frequency of such jumps.•Our study further characterized how RPA diffusion is slowed down by other RPA molecules through crowding. Replication Protein A (RPA) is asingle strandedDNA(ssDNA)binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations andis thought to modulate its function. Here, usingsingle moleculeconfocal fluorescencemicroscopy combinedwith optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA undertension.The diffusioncoefficientDis the highest (20,000nucleotides2/s) at 3pNtension and in 100 mMKCl and markedly decreases whentensionor salt concentrationincreases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes.