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.

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RNA quality control and timely termination of aberrant transcription are critical for functional gene expression. Here, we report that in Saccharomyces cerevisiae premature transcription termination of mRNAs is coordinated with the transcriptional elongation process and regulated by the evolutionarily conserved ATP-dependent chromatin remodeling complex INO80. Loss of INO80 sensitizes cells to the transcriptional elongation stress drug 6-azauracil and leads to enhanced pausing of elongating RNA Polymerase II across the genome. Transcriptional pausing positively correlates with premature termination of mRNA transcription and is pronounced proximally to promoters at sites of enhanced histone H3 binding to DNA. Cells with deficient INO80 complex accumulate short, unproductive mRNA transcripts on chromatin and are defective in transcription termination mediated by the Nrd1-Nab3-Sen1 (NNS) complex. We find that loss of INO80 compromises the interaction of the RNA surveillance factor Nab2 with short promoter-proximal mRNA transcripts. INO80 promotes cotranscriptional recruitment of Nab2 to chromatin by enabling its interaction with the histone variant H2A.Z. Finally, inactivation of the histone deacetylase complex Rpd3S/Rco1 reduces promoter-proximal pausing and enhances productive transcription through an NNS-dependent termination site when INO80 is compromised. Our work suggests that, by regulation of H2A.Zcontaining nucleosomes, INO80 orchestrates a mechanism for premature transcription termination, linking RNA quality control to the transcriptional process.

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