Ribodysgenesis: sudden genome instability in the yeast Saccharomyces cerevisiae arising from RNase H2 cleavage at genomic-embedded ribonucleotides
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PROVIDER: PRJNA819077 | ENA |
REPOSITORIES: ENA
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Project description:Yeast Saccharomyces cerevisiae has been widely used as a model system for studying genome instability. Here, heterozygous S. cerevisiae zygotes were generated to determine the genomic alterations induced by sudden introduction of active RNase H2. In combination of a custom SNP microarray, the patterns of chromosomal instability could be explored at a whole genome level. Ribonucleotides can be incorporated into DNA during replication by the replicative DNA polymerases. These aberrant DNA subunits are efficiently recognized and removed by Ribonucleotide Excision Repair, which is initiated by the heterotrimeric enzyme RNase H2. While RNase H2 is essential in higher eukaryotes, the yeast Saccharomyces cerevisiae can survive without RNase H2 enzyme, although the genome undergoes mutation, recombination and other genome instability events at an increased rate. Although RNase H2 can be considered as a protector of the genome from the deleterious events that can ensue from recognition and removal of embedded ribonucleotides, under conditions of high ribonucleotide incorporation and retention in the genome in a RNase H2-negative strain, sudden introduction of active RNase H2 causes massive DNA breaks and genome instability in a condition which we term “ribodysgenesis”. The DNA breaks and genome instability arise solely from RNase H2 cleavage directed to the ribonucleotide-containing genome. Survivors of ribodysgenesis have massive loss of heterozygosity events stemming from recombinogenic lesions on the ribonucleotide-containing DNA, with increases of over 1000X from wild-type. DNA breaks are produced over one to two divisions and subsequently cells adapt to RNase H2 and ribonucleotides in the genome and grow with normal levels of genome instability.
2022-03-26 | GSE199262 | GEO
Project description: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 (rnh1Δ), RNase H2 (rnh201Δ), or both RNase H1 and RNase H2 (rnh1Δ rnh201Δ). We found little effect on recombination in the rnh1Δ strain, but elevated recombination in both the rnh201Δ and the double-mutant strains; levels of recombination in the double mutant were about 50% higher than in the rnh201 single-mutant strain. An rnh201Δ mutant that additionally contained a mutation that reduces rNMP incorporation by DNA polymerase ε (pol2-M644L) had a level of instability similar to that observed in the presence of wild-type Polε. 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.
2015-09-23 | GSE73334 | GEO
Project description:Ribonucleotides can be incorporated into DNA during replication by the replicative DNA polymerases. These aberrant DNA subunits are efficiently recognized and removed by Ribonucleotide Excision Repair, which is initiated by the heterotrimeric enzyme RNase H2. While RNase H2 is essential in higher eukaryotes, the yeast Saccharomyces cerevisiae can survive without RNase H2 enzyme, although the genome undergoes mutation, recombination and other genome instability events at an increased rate. Although RNase H2 can be considered as a protector of the genome from the deleterious events that can ensue from recognition and removal of embedded ribonucleotides, under conditions of high ribonucleotide incorporation and retention in the genome in a RNase H2-negative strain, sudden introduction of active RNase H2 causes massive DNA breaks and genome instability in a condition which we term 'ribodysgenesis'. The DNA breaks and genome instability arise solely from RNase H2 cleavage directed to the ribonucleotide-containing genome. Survivors of ribodysgenesis have massive loss of heterozygosity events stemming from recombinogenic lesions on the ribonucleotide-containing DNA, with increases of over 1000X from wild-type. DNA breaks are produced over one to two divisions and subsequently cells adapt to RNase H2 and ribonucleotides in the genome and grow with normal levels of genome instability.
| S-EPMC9262587 | biostudies-literature
Project description: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 (rnh1Δ), RNase H2 (rnh201Δ), or both RNase H1 and RNase H2 (rnh1Δ rnh201Δ). We found little effect on recombination in the rnh1Δ strain, but elevated recombination in both the rnh201Δ and the double-mutant strains; levels of recombination in the double mutant were about 50% higher than in the rnh201 single-mutant strain. An rnh201Δ mutant that additionally contained a mutation that reduces rNMP incorporation by DNA polymerase ε (pol2-M644L) had a level of instability similar to that observed in the presence of wild-type Polε. 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. The details of these experiments are in press in Genetics This diploid S.cerevisiae strain used here is a hybrid between W303-1A and YJM789. The backgrounds are diverged by about 50,000 polymorphisms that are surveyed by custom CGH microarrays containing SNP-specific probes (St. Charles et al., 2012; Genetics 190: 1267-1284; PMID 22267500). The whole genome array surveyed loss of heterozygosity (LOH) events throughout the genome of in wild type, pol2-M644L, rnh1Δ, rnh201Δ, rnh201Δ pol2-M644L, and rnh1Δ rnh201Δ mutant backgrounds in 10, 12, 10, 19, 17, and 22 samples, respectively. The names of these strains are KO_198,KO_234, KO_73, KO_75, KO_244, and KO_5, respectively. Another assay was performed mapping reciprocal crossover events on the right arm of chromosome IV in the rnh201Δ (strains KO_135 and KO188) and the rnh1Δrnh201Δ (strain KO_132) mutant backgrounds. A reporter system with a wild type ADE2 on the end of chromosome IV allowed us to detect crossovers by seeing a red-white sectored colony. Each reciprocal event was mapped by doing a microarray specific for the right arm of chromosome IV (St. Charles and Petes, 2013; PLoS Genet. 1267-1284 PMCID PMC3616911) on the red and white sides of sectors. 21 sectors were analyzed for the rnh201Δ mutant while 14 were analyzed for the rnh1Δ rnh201Δ mutant. We mapped crossovers on the right arm of chromosome IV in the rnh201Δ mutant in four different diploid isolates: KO135_5 (1R/1W, 2R/2W, 4R/4W, 5R/5W, 12R/12W, and 13R/13W), KO_135_6 (1R/1W, 4R/4W, 5R/5W, 7R/7W, 9R/9W, 11R/11W, 13R/13W, and 17R/17W), KO 188_1 (1R/1W, and 4R/4W), and KO188_2 (6R/6W, 7R/7W, 11R/11W, and 12R/12W). We mapped crossovers on the right arm of chromosome IV in the rnh1Δ rnh201Δ mutant in two different diploid isolates: KO_132_31 (2R/2W, 6R/6W, 8R/8W, 15R/15W, 17R/17W, 21R/21W, 22R/22W, and 24R/24W) and KO_132_29 (3R/3W, 7R/7W, 18R/18W, 24R/24W, and 34R/34W).
2015-09-23 | E-GEOD-73334 | biostudies-arrayexpress
Project description:RNase H2 is a specialized enzyme that degrades RNA in RNA/DNA hybrids and deficiency of this enzyme causes a severe neuroinflammatory disease, Aicardi Goutières syndrome (AGS). However, the molecular mechanism underlying AGS is still unclear. Here, we show that RNase H2 is associated with a subset of genes, in a transcription-dependent manner where it interacts with RNA Polymerase II. RNase H2 depletion impairs transcription leading to accumulation of R-loops, structures that comprise RNA/DNA hybrids and a displaced DNA strand, mainly associated with short and intronless genes. Importantly, accumulated R-loops are processed by XPG and XPF endonucleases which leads to DNA damage and activation of the immune response, features associated with AGS. Consequently, we uncover a key role for RNase H2 in the transcription of human genes by maintaining R-loop homeostasis. Our results provide insight into the mechanistic contribution of R-loops to AGS pathogenesis.
2022-02-04 | GSE146970 | GEO
Project description:Class switch recombination generates antibody distinct isotypes critical to a robust adaptive immune system and defects are associated with auto-immune disorders and lymphomagenesis. Transcription is required during class switch to recruit the cytidine deaminase AID—an essential step for the formation of DNA doublestrand breaks—and strongly induces the formation of R loops within the immunoglobulin heavy chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here we report that cells lacking two enzymes involved in R loop removal— Senataxin and RNase H2—exhibit increased R loop formation and genome instability at the immunoglobulin heavy chain locus without impacting class switch recombination efficiency, transcriptional activity, or AID recruitment. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking Senataxin or RNase H2B alone. We propose that Senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis.
2022-12-14 | GSE201210 | GEO
Project description:R-loops represent a major source of replication stress but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest and restart in S. cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate normally their chromosomes under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Indeed, these cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative step in resuming replication. Similar impairments in nascent DNA resection at HU-arrested forks were observed in human cells lacking RNase H2. However, the addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation, fully restored fork resection. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.
2023-10-14 | GSE215896 | GEO
Project description:We report here the consequences of loss of Saccharomyces cerevisiae RNase H2 by comparing mRNA expression profiles in wild type yeast versus a strain lacking the RNH201 gene, encoding the catalytic subunit. Deleting RHN201 alters mRNA expression for hundreds of genes. Expression changes were based on seven biological replicates (3 from one batch and 4 from another batch) and are seen for many genes involved in stress responses and genome maintenance, consistent with a role of RNase H2 in removing ribonucleotides incorporated into DNA during replication. Differentially expressed genes include, those involved in ribosomal RNA processing and biogenesis and in tRNA modification, supports a role for RNase H2 in resolving R-loops formed during transcription of rRNA and tRNA. Other differentially expresses genes include putative or known helicases and nucleases maintenance of dsRNA viruses, which could be relevant to how mammalian RNase H2 dysfunction elicits an innate immune response leading to neurodegeneration.
2012-12-20 | GSE34668 | GEO
Project description:We report the application of high through-put tag sequencing to measure the location and strand of DNA embedded ribonucleotides in the yeast genome. Mutations in the catalytic subunits of the polymerases (pol1-L868M, pol2-M644G and pol3-L612M) lead to the increased incorporation of ribonucleotides during DNA replication, providing an in vivo label with which to track the contribution of each polymerase to the fully replicated genome. Yeast strains used in this study are deleted for rnh201, encoding the catalytic subunit of the RNase H2 gene so that embedded ribonucleotides are not rapidly removed by ribonucleotide excision repair following DNA replication. Analysis of this data demonstrates that polymerase alpha contributes to the fully replicated genome. Sequencing of DNA embedded ribonucleotides in S. cerevisiae strains to map the contribution of replicative polymerases to the fully replicated genome.
2015-01-26 | E-GEOD-64521 | biostudies-arrayexpress
Project description:We report here the consequences of loss of Saccharomyces cerevisiae RNase H2 by comparing mRNA expression profiles in wild type yeast versus a strain lacking the RNH201 gene, encoding the catalytic subunit. Deleting RHN201 alters mRNA expression for hundreds of genes. Expression changes were based on seven biological replicates (3 from one batch and 4 from another batch) and are seen for many genes involved in stress responses and genome maintenance, consistent with a role of RNase H2 in removing ribonucleotides incorporated into DNA during replication. Differentially expressed genes include, those involved in ribosomal RNA processing and biogenesis and in tRNA modification, supports a role for RNase H2 in resolving R-loops formed during transcription of rRNA and tRNA. Other differentially expresses genes include putative or known helicases and nucleases maintenance of dsRNA viruses, which could be relevant to how mammalian RNase H2 dysfunction elicits an innate immune response leading to neurodegeneration. We examine the consequences of loss of Saccharomyces cerevisiae RNase H2 by comparing mRNA expression profiles in wild type yeast versus a strain lacking the RNH201 gene encoding the catalytic subunit. Expression changes were based on seven biological replicates (3 from one batch and 4 from another batch). For strain reference please see S.A. Nick McElhinny, et al, Genome instability due to ribonucleotide incorporation into DNA, Nat Chem Bio 6, 774-781 (2010).
2012-12-20 | E-GEOD-34668 | biostudies-arrayexpress