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

Abstract

Cells carrying deletions of genes encoding H-class ribonucleases display elevated rates of chromosome instability. The role of these enzymes is to remove RNA-DNA associations including persistent mRNA-DNA hybrids (R-loops) formed during transcription, and ribonucleotides incorporated into DNA during replication. RNases H1 and H2 can degrade the RNA component of R-loops, but only RNase H2 can initiate accurate ribonucleotide excision repair (RER). In order to examine the specific contributions of these activities to chromosome stability, we measured rates of loss-of-heterozygosity (LOH) in diploid Saccharomyces cerevisiae yeast strains carrying the rnh201-RED separation-of-function allele, encoding a version of RNase H2 that is RER-defective, but partly retains its other activity. The LOH rate in rnh201-RED was intermediate between RNH201 and rnh201Δ. In strains carrying a mutant version of DNA polymerase ε (pol2-M644G) that incorporates more ribonucleotides than normal, the LOH rate in rnh201-RED was as high as the rate measured in rnh201Δ. Topoisomerase 1 cleavage at sites of ribonucleotide incorporation has been recently shown to produce DNA double strand breaks. Accordingly, in both the POL2 and pol2-M644G backgrounds, the LOH elevation in rnh201-RED was suppressed by top1Δ. In contrast, in strains that incorporate fewer ribonucleotides (pol2-M644L) the LOH rate in rnh201-RED was low and independent of topoisomerase 1. These results suggest that both R-loop removal and RER contribute substantially to chromosome stability, and that their relative contributions may be variable across different regions of the genome. In this scenario, a prominent contribution of R-loop removal may be expected at highly transcribed regions, whereas RER may play a greater role at hotspots of ribonucleotide incorporation.

Keywords: Loss-of-heterozygosity (LOH); R-loops; RNH201; RNase H2; Ribonucleotides.

Figure 1. Substrates of H-Class RNases, experimental system, and LOH rate analysis

(A) Schematic representation of the substrate specificity of H-Class RNases. The black arrow lines link the various enzymes to their RNA-DNA substrates. The gray dashed arrow line indicates that evidence of topoisomerase 1 cleavage at ribonucleotides has only been observed in the absence of functional RER. (B) Depiction of the hemizygous chromosomal configuration used in the LOH assay. One of the homologs of Chr7 (dark green) has an insertion of the counter-selectable CORE2 cassette (_KlURA3-_ScURA3-KanMX4) [24] downstream of the MAL13 gene (distal side), ~20 kb from the right telomere (7R); the second homolog (light green) does not. A DNA lesion on the right arm of the dark green homolog may initiate an allelic mitotic recombination event leading to LOH, making the distal portion of the chromosome homozygous for the light green DNA sequence, and thus rendering that cell resistant to 5-FOA. (C) Quantitative analysis of LOH. The columns represent the median Chr7 right arm LOH rate for each genotype, and the error bars represent 95% confidence intervals (CI). The same data are presented in numeric form in Table S2, and statistical significance of pairwise comparisons are available in Table S3. All genotypes indicated in the X-axis are homozygous in the experimental diploids. The greater than (>) symbol indicates that the Y-axis was cropped to save space and to facilitate visualization of differences between the lower rates. The upper 95% CI limit of the pol2-M644G rnh1Δ rnh201-RED TOP1 LOH rate was 182.48 ×2×10⁻⁵/cell/division. Intentional gaps were left in the data columns for the POL2 rnh1Δ rnh201Δ top1Δ and the pol2-M644G rnh1Δ rnh201Δ TOP1 genotypes to emphasize the fact that these triple mutant combinations are synthetic lethal as reported previously [–35].