The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions.

Charanya Kumar,Sahil Batra,Dirk Remus,Jack D Griffith

doi:10.7554/elife.72286

Charanya Kumar, Sahil Batra + Show 2 more

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https://doi.org/10.7554/elife.72286

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Abstract

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

Highlights

Genome maintenance is dependent on the complete and accurate replication of the chromosomal DNA prior to cell division
Here, we employ the reconstituted budding yeast DNA replication system in combination with purified R-loop templates to study orientation-specific R-loop-replisome collisions (Devbhandari et al, 2017; Devbhandari and Remus, 2020; Yeeles et al, 2015). We demonstrate that both CD and HO R-loops can adversely affect fork progression, dependent on the specific configuration of RNA:DNA hybrids and G4s
R-loops have been widely recognized as a major determinant of genome instability caused by transcription-replication conflict (TRC), the mechanism by which R-loops impede DNA replication has remained obscure

Summary

Introduction

Genome maintenance is dependent on the complete and accurate replication of the chromosomal DNA prior to cell division. The latter may include enhanced RNAP stalling (Belotserkovskii et al, 2010), establishment of repressive chromatin structures (Castellano-Pozo et al, 2013; Garcia-P ichardo et al, 2017; Garcia-Rubio et al, 2018), or induction of DNA breaks by nucleases (Sollier et al, 2014; Su and Freudenreich, 2017) To overcome this limitation, here, we employ the reconstituted budding yeast DNA replication system in combination with purified R-loop templates to study orientation-specific R-loop-replisome collisions (Devbhandari et al, 2017; Devbhandari and Remus, 2020; Yeeles et al, 2015). Our data reveals how the specific structure of R-loops determines the outcome of R-loop-d ependent TRC

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Discussion

Materials and methods

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