title: 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.
authors.affiliation: Department of Environmental and Radiological Health Sciences and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Cornelio
authors.affiliation: Department of Environmental and Radiological Health Sciences and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO 80523, USA; Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Hailey N C
authors.initials: HN
authors.lastname: Sedam
authors.affiliation: Department of Environmental and Radiological Health Sciences and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Ferrarezi
authors.affiliation: Department of Environmental and Radiological Health Sciences and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO 80523, USA; Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Nadia M V
authors.initials: NM
authors.lastname: Sampaio
authors.affiliation: Department of Environmental and Radiological Health Sciences and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO 80523, USA; Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: lucas.argueso@colostate.edu.
authors.forename: Juan Lucas
authors.initials: JL
authors.lastname: Argueso
mesh_terms: D043171:Chromosomal Instability; D004257:DNA Polymerase II / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism; D019656:Loss of Heterozygosity; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D004271:DNA, Fungal; D012331:RNA, Fungal; D012333:RNA, Messenger; D012265:Ribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D004257:DNA Polymerase II; D012260:Ribonucleases; C569437:Rnh201 protein, S cerevisiae; D004264:DNA Topoisomerases, Type I
keywords: Loss-of-heterozygosity (LOH); R-loops; RNH201; RNase H2; Ribonucleotides