Elevated Genome-Wide Instability in Yeast Mutants Lacking RNase H Activity

Two types of RNA: DNA associations can lead to genome instability: the formation of R-loops during transcription and the incorporation of ribonucleotide monophosphates (rNMPs) into DNA during replication. Both ribonuclease (RNase) H1 and RNase H2 degrade the RNA component of R-loops, whereas only RNase H2 can remove one or a few rNMPs from DNA. We performed high-resolution mapping of mitotic recombination events throughout the yeast genome in diploid strains of Saccharomyces cerevisiae lacking RNase H1 (rnh1 Delta), RNase H2 (rnh201 Delta), or both RNase H1 and RNase H2 (rnh1 Delta rnh201D). We found little effect on recombination in the rnh1D strain, but elevated recombination in both the rnh201 Delta and the double-mutant strains; levels of recombination in the double mutant were similar to 50% higher than in the rnh201 single-mutant strain. An rnh201 Delta mutant that additionally contained a mutation that reduces rNMP incorporation by DNA polymerase epsilon (pol2-M644L) had a level of instability similar to that observed in the presence of wild-type Pol epsilon. This result suggests that the elevated recombination observed in the absence of only RNase H2 is primarily a consequence of R-loops rather than misincorporated rNMPs.