Differential roles of the RNases H in preventing chromosome instability

Abstract

DNA:RNA hybrids can lead to DNA damage and genome instability. This damage can be prevented by degradation of the RNA in the hybrid by two evolutionarily conserved enzymes, RNase H1 and H2. Indeed, RNase H-deficient cells have increased chromosomal rearrangements. However, the quantitative and spatial contributions of the individual enzymes to hybrid removal have been unclear. Additionally, RNase H2 can remove single ribonucleotides misincorporated into DNA during replication. The relative contribution of DNA:RNA hybrids and misincorporated ribonucleotides to chromosome instability also was uncertain. To address these issues, we studied the frequency and location of loss-ofheterozygosity (LOH) events on chromosome III in Saccharomyces cerevisiae strains that were defective for RNase H1, H2, or both. We showed that RNase H2 plays the major role in preventing chromosome III instability through its hybrid-removal activity. Furthermore, RNase H2 acts pervasively at many hybrids along the chromosome. In contrast, RNase H1 acts to prevent LOH within a small region of chromosome III where the instability is dependent upon two hybrid-prone sequences. This restriction of RNase H1 activity to a subset of hybrids is not the result of its constrained localization, because we found it at hybrids genome-wide. This result suggests that the genome-protection activity of RNase H1 is regulated at a step after hybrid recognition. The global function of RNase H2 and the region-specific function of RNase H1 provide insight into why these enzymes with overlapping hybrid-removal activities have been conserved throughout evolution.RNase H | R-loops | DNA:RNA hybrids | chromosome instability | genome instability P reventing chromosome instability is an essential process for maintaining genetic information. A source of chromosome instability is the accumulation of R-loops, which form when an RNA molecule hybridizes with a portion of genomic DNA, creating a DNA:RNA hybrid and a displaced single-stranded DNA (reviewed in ref. 1). One mechanism to prevent hybridmediated damage involves RNase H1 and RNase H2, two endogenous enzymes, conserved from bacteria to humans, that can degrade the RNA in R-loops (reviewed in ref.2). RNase H2 also functions in the removal of single ribonucleotides that are inappropriately incorporated into DNA by DNA polymerases during replication. Why RNase H1 and H2, which appear to have overlapping functions, remain highly conserved across many branches of life has been an outstanding question. Two areas of inquiry that will help address this conundrum are (i) does one of these RNases carry the major burden of preventing spontaneous R-loop-mediated chromosome instability, and, if so which, and (ii) do RNase H1 and H2 protect the same or different regions of the genome from R-loop-mediated damage?Whether RNase H1 and H2 contribute differentially to protecting against hybrid-mediated genome instability has been controversial. Studies of the inactivation of RNase H1 in yeast have shown little effect on chromosome insta...

Both R-loop removal and ribonucleotide excision repair activities of RNase H2 contribute substantially to chromosome stability

“…These results align well with those reported recently for a Chr3 instability assay that detects LOH within a relatively narrow region (98 Kb) plus Chr3 monosomy, each class accounting for about half of the overall instability rate [4]. However, that study did not report chromosome instability rates for the RNH1 rnh201-RED single mutant, and therefore was unable fully contextualize the difference between the chromosome stabilization contributed by the RER and tandem rNMP/R-loop removal activities of RNase H2, versus the contributions of RER and the combined tandem rNMP/R-loop removal activities from RNase H1 and H2.…”

“…Thus, loss of RNase H1 by itself should have a limited impact on chromosome instability. Consistently, the rate of Chr7 LOH in the rnh1Δ single mutant did not significantly change relative to wild type (p=0.8133), but increased by twelve fold in the rnh1Δ rnh201Δ double (p<0.0001), in agreement with previous reports [4, 18]. This pronounced elevation in LOH when both RNase H’s are absent underscores how important tandem rNMP/R-loop removal is for chromosome stabilization as a whole.…”

“…1C). While in principle RNases H1 and H2 both have redundant tandem rNMP and R-loop removal activities, RNase H2’s activity is thought to be more prevalent, having a global genomic role, whereas RNase H1’s role might be confined to a subset of R-loop forming regions [4]. Thus, loss of RNase H1 by itself should have a limited impact on chromosome instability.…”

“…1A). RNases H1 and H2 have a redundant role in degrading the RNA component of RNA-DNA hybrids and tandem rNMPs in DNA, with RNase H2 likely playing a more prevalent role [4]. In addition, RNase H2, but not RNase H1, has a second distinct activity, which is to initiate the accurate removal of single rNMPs incorporated into DNA (ribonucleotide excision repair - RER) [5].…”

“…1A). Two recent studies [4, 20] reported mildly elevated chromosome instability phenotypes in rnh201-RED compared to wild type, but the elevation was not as high as that seen in rnh201Δ . In each case the authors concluded that RER was not a substantial contributor to chromosome stability.…”