Rapid long-distance migration of RPA on single stranded DNA occurs through intersegmental transfer utilizing multivalent interactions

Replication Protein A (RPA) is a single stranded DNA (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 and is thought to modulate its function. Here, using single molecule confocal fluorescence microscopy combined with optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA under tension. The diffusion coefficient D is the highest (20,000 nucleotides2/s) at 3 pN tension and in 100 mM KCl and markedly decreases when tension or salt concentration increases. 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. SignificanceReplication Protein A (RPA) binds to the exposed single stranded DNA (ssDNA) during DNA metabolism. RPA dynamics are essential to reposition RPA on ssDNA and recruit downstream proteins at the bound site. Here in this work, we perform a detailed biophysical study on dynamics of yeast RPA on ssDNA. We show that RPA can diffuse on ssDNA and is affected by tension and salt. Our observations are best explained by the intersegmental transfer model where RPA can transiently bridge two distant DNA segments for its migration over long distances. We further dissect the contributions of the trimerization core of RPA and other adjacent RPA molecules on RPA migration. This study provides detailed experimental and computational insights into RPA dynamics on ssDNA.