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bioRxiv · 10.1101/2022.09.30.510372

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

Mersch, K.·Sokoloski, J. E.·Nguyen, B.·Galletto, R.·Lohman, T. M.
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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.

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Mersch, K., Sokoloski, J. E., Nguyen, B., Galletto, R., Lohman, T. M.. 2022-10-01. Helicase Activity Promoted Through Dynamic Interactions Between a ssDNA Translocase and a Diffusing SSB Protein. https://doi.org/10.1101/2022.09.30.510372

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