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

Abstract

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...

Differential roles of the RNases H in preventing chromosome instability

“…The conclusion that RNase H2, but not H1, carries the major load of protecting cells against large-scale LOH corroborates a previous study (4). 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.…”

“…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).…”

“…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.…”

Global analysis of genomic instability caused by DNA replication stress in Saccharomyces cerevisiae

“…Because it takes about 25 divisions to form a colony from a single cell, the rates of alterations per cell division in the strains with low levels of DNA polymerase α and δ are about 0.6 and 0.3, respectively. By a similar calculation using the data of O'Connell et al (17), the rate of genetic alterations in wild-type cells is about 2.4 × 10 −3 per division. Thus, low levels of either DNA polymerase α or δ elevate genomic instability by about two orders of magnitude.…”

RNase H enables efficient repair of R-loop induced DNA damage

“…However, a number of observations support our hypothesis of R-loops as inhibitors of repair. Inactivation of RNase H2 (rnh201D) by itself leads to large increases of genomic hybrids and loss of heterozygosity compared to wild-type (Wahba et al, 2016;O'Connell et al, 2015). This result suggests that loss of RNase H2 generates elevated levels of DNA damage.…”