“Helicase” Activity Promoted Through Dynamic Interactions Between a ssDNA Translocase and a Diffusing SSB Protein

Abstract

Replication protein A (RPA) is a eukaryotic single stranded (ss) DNA binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches we show that S. cerevisiae Pif1 can use its ATP-dependent 5 to 3 translocase activity to chemo-mechanically push a single human RPA (hRPA) directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and demonstrate a new mechanism by which directional DNA unwinding can be achieved through the combined action of a ssDNA translocase that pushes an SSB protein.

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

“…In the absence of force, DBDs of RPA can bind distant sites at the same time and effectively stabilize the condensed DNA (65). Recent study on human RPA did not observe a significant influence of force on its diffusion (53). Our study on yeast RPA showed a clear force dependence on the diffusion of the protein both in experiment and simulations.…”

“…Although the effect of DNA tension on SSB diffusion on DNA has been studied previously (51), diffusive property of yeast RPA on ssDNA longer than 120 oligonucleotides length, or under mechanical tension, has not been reported yet. A recent study by Mersch et al have investigated diffusion of human RPA (hRPA) on 20 kbp long ssDNA and reported that the hRPA diffusion does not depend significantly on tension or salt concentration (53).…”