Stimulation of Chromosomal Rearrangements by Ribonucleotides

Conover, Hailey N.; Lujan, Scott A.; Chapman, Mary J.; Cornélio, Deborah Afonso; Sharif, Rabab; Williams, Jessica S.; Clark, Allan; Camilo, Francheska; Kunkel, Thomas A.; Argueso, Juan Lucas

doi:10.1534/genetics.115.181149

Cited by 45 publications

(68 citation statements)

References 38 publications

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“…Although our work and that of O'Connell and colleagues (4) concluded that this protection results from RNase H2's removing R-loops, a different study of RNase H2's activities by Conover and colleagues (5) suggested that this protection is afforded by RNase H2's ability to remove misincorporated nucleotides. In fact, many studies have shown that misincorporated ribonucleotides can lead to DNA damage and mutation in a topoisomerase I-dependent manner (13)(14)(15)(16).…”

Section: Discussionmentioning

confidence: 49%

“…Studies of the inactivation of RNase H1 in yeast have shown little effect on chromosome instability (3,4), whereas inactivation of RNase H2 has been shown to increase chromosome instability (4,5). However, results from the Conover study (5) suggested that the elevated instability in the RNase H2-deficient cells is caused by elevated misincorporation of ribonucleotides rather than by the failure to remove hybrids. In contrast, results from the O'Connell study (4) suggested the opposite: that the elevated instability is caused by hybrids rather than by misincorporated ribonucleotides.…”

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confidence: 99%

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Differential roles of the RNases H in preventing chromosome instability

Zimmer

Koshland

2016

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

confidence: 49%

mentioning

confidence: 99%

Differential roles of the RNases H in preventing chromosome instability

Zimmer

Koshland

2016

Proc. Natl. Acad. Sci. U.S.A.

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Section: Discussionmentioning

confidence: 93%

“…Although we did observe a small reduction in LOH in the rnh201D pol2-M644L strain relative to the rnh201D strain in one of our LOH assays, the reduction was only 20%. In a similar assay, the rate of LOH in an rnh201D strain was reduced less than twofold by the pol2-M644L mutation (Conover et al 2015).Based on subtle effect of the pol2-M644L mutation, we suggest that most of the LOH in the rnh201D mutant reflects persistent R-loops rather than persistent rNMPs. Our data indicate that RNase H2 can remove most, if not all, of the R-loops that accumulate in the absence of RNase H1, but that RNase H1 can remove only a relatively small fraction of the Figure 6 Classes of red/white sectors resulting from RCO.…”

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confidence: 76%

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

O’Connell¹,

Jinks-Robertson

Petes

2015

Genetics

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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 highresolution mapping of mitotic recombination events throughout the yeast genome in diploid strains of Saccharomyces cerevisiae lacking RNase H1 (rnh1D), RNase H2 (rnh201D), or both RNase H1 and RNase H2 (rnh1D rnh201D). We found little effect on recombination in the rnh1D strain, but elevated recombination in both the rnh201D and the double-mutant strains; levels of recombination in the double mutant were 50% higher than in the rnh201 single-mutant strain. An rnh201D mutant that additionally contained a mutation that reduces rNMP incorporation by DNA polymerase e (pol2-M644L) had a level of instability similar to that observed in the presence of wild-type Pol e. 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.KEYWORDS RNase H1; RNase H2; loss of heterozygosity; mitotic recombination; microarrays R NA transcripts can stably associate with the DNA template during transcription, giving rise to R-loop structures containing an RNA:DNA hybrid and an unpaired DNA strand. Such R-loops can stall DNA replication forks and are a major source of recombination events that are stimulated by high levels of transcription (reviewed by Aguilera and Garcia-Muse 2012). Transcription-associated recombination (TAR) is generally strongest when the transcription machinery and a replication fork approach each other head on (Prado and Aguilera 2005), but there is also an orientation-independent component to such conflicts ). Importantly, TAR is highly elevated when RNA processing is perturbed and R-loops accumulate, or when R-loop removal mechanisms are disabled. The RNA component of R-loops can be removed by the Sen1 RNA-DNA helicase (Mischo et al. 2011) or degraded by ribonuclease (RNase) H1 or RNase H2 (reviewed in Cerritelli and Crouch 2009), which are generally considered to be functionally redundant.R-loops in yeast have been quantified genome-wide using immunoprecipitation with an antibody specific to DNA:RNA hybrids, followed by hybridization to a microarray or by DNA sequencing. This type of analysis has been done in wild type (Chan et al. 2014;El Hage et al. 2014), mRNA processing defective (Chan et al. 2014), RNase H-defective (Chan et al. 2014;El Hage et al. 2014), and RNA-DNA helicase (Sen1)-defective strains (Chan et al. 2014). In wild-type strains, Chan et al. (2014) observed RNA-DNA hybrid accumulation at Ty retrotransposons, near telomeres, within the ribosomal RNA (rRNA) gene cluster, and at highly transcribed GC-rich genes. El Hage et al. (2014) found hybrid accumulation in wild-type strains at highly transcribed genes, within the rRNA gen...

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“…Somewhat surprisingly, RNA/DNA hybrids were found at higher than normal levels in cell lines from AGS patients with mutations in RNASEH2A , RNASEH2B , SAMHD1 and TREX1 , suggesting hybrids as possible causes of AGS[83]. The debate between consequences of rNMPs in DNA and failure to resolve RNA/DNA hybrids continues.. Two recent studies in S. cerevisiae examining recombination in diploid strains reported genome instability in the absence of RNase H2 but reached opposite conclusions as to the contributions of the two RNase H2 activities to the observed instability [59, 84]. Clearly more work lies ahead that will be aided by a RNase H2-RED (Ribonucleotide Excision Defective) mutant that lacks RER but can hydrolyze RNA/DNA hybrids[85].…”

Section: Ribonucleotides In Dna Of Aicardi Goutières Syndrome (Ags) Amentioning

confidence: 99%

The Balancing Act of Ribonucleotides in DNA

Cerritelli

Crouch

2016

Trends in Biochemical Sciences

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The abundance of ribonucleotides in DNA remained undetected until recently because they are efficiently removed by the Ribonucleotides Excision Repair pathway, a process similar to Okazaki fragment processing after incision by RNase H2. All DNA polymerases incorporate ribonucleotides during DNA synthesis. How many, when and why they are incorporated has been the focus of intense work during recent years by many labs. In this review, we discuss recent advances in ribonucleotide incorporation by eukaryotic DNA polymerases that suggest an evolutionarily conserved role for ribonucleotides in DNA and review the data that indicate that removal of ribonucleotides plays an important role in maintaining genome stability.

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Stimulation of Chromosomal Rearrangements by Ribonucleotides

Cited by 45 publications

References 38 publications

Differential roles of the RNases H in preventing chromosome instability

Differential roles of the RNases H in preventing chromosome instability

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

The Balancing Act of Ribonucleotides in DNA

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