Transient RNA-DNA Hybrids Are Required for Efficient Double-Strand Break Repair

Abstract

RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNase H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-strand break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous-recombination (HR)-mediated DSB repair process and that RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability.

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast

“…For instance, contrary to what was proposed before [16,23], we could not detect significant R-loop formation either over heterochromatin repeats at centromeres or over gene terminator regions in fission yeast. Although we confirmed that the S9.6 antibody immuno-precipitates RNA species associated with centromeric repeats and terminator regions (Figs.…”

“…The first map was generated by DRIP-seq and as a result was not stranded and had low resolution [16]. The second map was made using a ChIP-exo approach to increase resolution, but it only provided a map of R-loops in cells lacking RNase H activity ( rnh1Δrnh201Δ cells) and the sensitivity of the signals to RNase H was not controlled [23]. The maps produced here are stranded and have high resolution, and the sensitivity of the signals to in vivo RNase H level manipulations has been validated.…”

Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids

“…6a). Considering that RNA-DNA hybrids have recently been shown to form at DNA breaks, Polθ-helicase dissociation of these structures may also contribute to DSB repair (27).…”

“…For example, R-loops have long been associated with replicative stress and genome instability, whereas more recent work indicates that RNA-DNA hybrids can also promote DNA repair by mechanisms that remain to be elucidated (23)(24)(25)(26)(27). Considering the importance of RNA-DNA structures in DNA repair and genome instability, we proceeded to examine whether Polθ-helicase unwinds RNA-DNA duplexes with similar efficiency.…”

Chromatin modifiers and remodellers in DNA repair and signalling

“…As discussed in this review, emerging evidence indicates that there is a repair process that occurs in regions open for transcription interfacing transcriptional arrest and repair, akin to the distinction between transcription coupled repair and global genome repair at UV photoproducts. An emerging topic in the field of DNA repair is the contribution of transcription and RNA to the repair process [34][35][36]. In particular, DNA-RNA hybrids or R-loops can present a source of DNA damage but equally can profoundly influence the repair process.…”