A Saccharomyces cerevisiae RNase H2 Interaction Network Functions To Suppress Genome Instability

Abstract

Errors during DNA replication are one likely cause of gross chromosomal rearrangements (GCRs). Here, we analyze the role of RNase H2, which functions to process Okazaki fragments, degrade transcription intermediates, and repair misincorporated ribonucleotides, in preventing genome instability. The results demonstrate that rnh203 mutations result in a weak mutator phenotype and cause growth defects and synergistic increases in GCR rates when combined with mutations affecting other DNA metabolism pathways, including homologous recombination (HR), sister chromatid HR, resolution of branched HR intermediates, postreplication repair, sumoylation in response to DNA damage, and chromatin assembly. In some cases, a mutation in RAD51 or TOP1 suppressed the increased GCR rates and/or the growth defects of rnh203⌬ double mutants. This analysis suggests that cells with RNase H2 defects have increased levels of DNA damage and depend on other pathways of DNA metabolism to overcome the deleterious effects of this DNA damage.

Stimulation of Chromosomal Rearrangements by Ribonucleotides

“…Although the phenotypic differential provided by this assay system is small, the highest rate of NAHR was measured in the rnh201D pol2-M644G strain, and chromosomal size polymorphisms other than the selected translocations were only detected in the rnh201D background. Together, these observations were consistent with earlier reports for a role of ribonucleotides in the generation of gross chromosomal rearrangements in yeast (Wahba et al 2011;Allen-Soltero et al 2014) and increased cytogenetic abnormalities in mammalian cells (Reijns et al 2012).…”

“…Although the phenotypic differential provided by this assay system is small, the highest rate of NAHR was measured in the rnh201D pol2-M644G strain, and chromosomal size polymorphisms other than the selected translocations were only detected in the rnh201D background. Together, these observations were consistent with earlier reports for a role of ribonucleotides in the generation of gross chromosomal rearrangements in yeast (Wahba et al 2011;Allen-Soltero et al 2014) and increased cytogenetic abnormalities in mammalian cells (Reijns et al 2012).The recombinogenic effects associated with RNase H2 mutants may result from misprocessing of scattered ribonucleotides incorporated into DNA, misprocessing of R-loops, or a combination of these two defects. The observation that increased and decreased ribonucleotide incorporation by Pol e correlates with the LOH rate leads us to propose that much of the LOH observed here in RNase H2 mutants is triggered by ribonucleotides incorporated by Pol e during leading strand replication.…”

“…Interestingly, the mutagenic effect of ribonucleotides appears to be asymmetric, according to which DNA polymerase was responsible for their incorporation. Specifically, TOP1-dependent 2-to 5-bp deletions in an RNase H2 mutant background were shown to be the product of ribonucleotides incorporated by Pol e, but not by Pol a and Pol d (Williams et al 2015).Earlier reports described increased chromosome-scale instability in RNase H2 mutant haploid yeast cells, including TOP1-dependent gene conversion (Potenski et al 2014) and gross chromosomal rearrangements (Allen-Soltero et al 2014). Another study measured the rate of LOH at Chr3 in RNase H homozygous mutant diploids (Wahba et al 2011).…”

“…In addition to the point mutations mentioned above, larger types of genome instability have also been observed in RNase H2-defective cells. For example, in a study of gross chromosomal rearrangements (GCRs) in haploid yeast cells, RNase H2 defects alone had little effect, but GCR rates were elevated in double mutant strains lacking the noncatalytic Rnh203 subunit in combination with deletions of any of eight other genes affecting DNA metabolism (Allen-Soltero et al 2014). An earlier GCR study reported that rnh201D single mutants displayed a fourfold increase in instability of a nonessential yeast artificial chromosome (YAC loss and terminal deletions) (Wahba et al 2011).…”

Roles of DNA helicases and Exo1 in the avoidance of mutations induced by Top1-mediated cleavage at ribonucleotides in DNA

“…Consistent with the idea that the slippage events in the dinucleotide reporter arise as a result of Top1 cleavage at rNMP residues, the rnh202D sgs1D slippage events were mostly eliminated by the top1D mutation, being reduced from 140-fold increase over wildtype to 8X increase. A synergistic interaction between sgs1D and rnh203D (which has another subunit of RNaseH2 deleted) on gross chromosome rearrangement (GCR) rates has been reported, 15 however in this published study the synergistic increase was independent of Top1.…”