RNase H enables efficient repair of R-loop induced DNA damage.

Jeremy D Amon,Douglas Koshland

doi:10.7554/elife.20533

Abstract

R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. We present a model that the persistence of R-loops at sites of DNA damage induces repair by break-induced replication (BIR). This R-loop induced BIR is particularly susceptible to the formation of lethal repair intermediates at the rDNA because of a barrier imposed by RNA polymerase I.

Highlights

R-loops are structures that form when RNA invades double-stranded DNA and hybridizes to complementary genomic sequences (Gaillard and Aguilera, 2016)
To better understand the mechanisms by which DNA:RNA hybrids contribute to genome instability, we began by characterizing DNA damage in exponentially dividing wild-type, rnh1D, rnh201D, and rnh1D rnh201D budding yeast cells
As we suggested for double mutants, the accumulation of DNA damage after bulk S-phase was consistent with double-stranded breaks (DSBs) being formed by impaired late-firing replication forks, such as those seen at the ribosomal DNA locus (rDNA)

Summary

Introduction

R-loops are structures that form when RNA invades double-stranded DNA and hybridizes to complementary genomic sequences (Gaillard and Aguilera, 2016). R-loops can form spontaneously across many genomic loci, but the activity of two endogenous RNases H prevents their accumulation and persistence (Cerritelli and Crouch, 2009). Efforts to map R-loops genome-wide have shown that in the absence of RNase H activity, the levels of hybrids formed at susceptible loci increase dramatically (Wahba et al, 2016). This increase in hybrids is associated with increased rates of genome instability that include loss of heterozygosity (LOH) events, loss of entire chromosomes, and recombination at the ribosomal locus (Wahba et al, 2011; O’Connell et al, 2015). The RNases H have been implicated as important protectors of genome stability

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