"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 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 RPA from an 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 RPA such that it can 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 an ssDNA translocase that pushes an SSB protein. This work was supported by National Institutes of Health Grants 5R35GM136632 (to T.M.L.) and 1R35GM139508 (to R.G.) and American Cancer Society Grant PF-15-040-01-DMC (to J.E.S) and NIH Instrumentation Grant S10OD030315.