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

Grant D Schauer,Lisanne M Spenkelink,Jacob S Lewis,Olga Yurieva,Stefan H Mueller,Antoine M Van Oijen,Michael E O’Donnell

doi:10.1073/pnas.2020189117

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.

Highlights

Efficient and faithful replication of the genome is essential to maintain genome stability
The guide RNA (gRNA) and dead” Cas9 (dCas9) were preincubated with the template DNA before addition of CMG
R-loops are thought to occur at transfer RNA gene clusters and other highly transcribed genes in vivo

Summary

Introduction

Efficient and faithful replication of the genome is essential to maintain genome stability. There are numerous obstacles to progression of the replisome during the process of chromosome duplication These obstacles include RNA-DNA hybrids (R-loops), DNA secondary structures, transcribing RNA polymerases, and other tightly bound proteins [5,6,7,8,9]. Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. The dCas9–gRNA complex forms a roadblock consisting of an R-loop and a tightly bound protein (dCas9), a construct that is similar to a stalled RNA polymerase This roadblock (hereafter dCas R-loop) arrests replisomes independent of whether the dCas R-loop is targeted to the leading or lagging strand [30]. Use of monomeric helicases may have evolved to aid replisome bypass of protein-DNA and protein-bound R-loop blocks

Methods

Results

Conclusion