Replisome bypass of a protein-based R-loop block by Pif1.

Proceedings of the National Academy of Sciences of the United States of America
Grant D SchauerMichael E O'Donnell

Abstract

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5'-3' direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

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Citations

Jan 24, 2021·Molecular Cell·Kyle B VrtisJohannes C Walter
Jun 15, 2021·Frontiers in Cell and Developmental Biology·Maksym Shyian, David Shore
Aug 19, 2021·Critical Reviews in Biochemistry and Molecular Biology·Amy J Fernandez, James M Berger
Aug 17, 2021·DNA Repair·Samira KemihaPhilippe Pasero
Nov 12, 2021·Protein Science : a Publication of the Protein Society·Alicia K ByrdKevin D Raney

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Methods Mentioned

BETA
immunoprecipitation
pull-down
fluorescence imaging
electrophoresis

Software Mentioned

ImageJ
Chroma
Fiji
Matlab

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