Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability

Lisa Prendergast,Rolando Berlinguer-Palmini,Daniel Rico,Jonathan M G Higgins,Philippe Pasero,Sarah Greener,Inmaculada Hernandez,Rossitsa Hristova,Anastas Gospodinov,Urszula L Mcclurg,Katie Veitch,Manolis Papamichos-Chronakis

doi:10.1038/s41467-020-18306-x

Abstract

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.

Highlights

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells
We questioned whether the role of INO80 in promoting DNA replication is dependent on transcription. siControl and siINO80 PC3 cells were treated with the transcriptional inhibitors α-amanitin or cordycepin and analysed for DNA synthesis rates by CldU/IdU DNA fibre pulse labelling assay (Fig. 1a)
We elucidate a role of the human INO80 complex in DNA replication

Summary

Introduction

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. We show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replicationassociated DNA damage in cancer cells. Counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, enabling unlimited proliferation in cancers. Recent studies indicate that R-loops are highly abundant and induce replication stress in cancer cells[6,7] This raises the question how cancer cells sustain sufficient DNA synthesis rates in the presence of increased transcription-replication conflicts. The histone chaperone complex FACT, which promotes nucleosomal integrity[10] and facilitates transcription in the presence of chromatin[11], prevents R-loop accumulation and promotes resolution of transcriptionreplication conflicts[12]. The role of INO80 in cancer cell proliferation remains largely elusive

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Results

Conclusion