Epistasis analysis between homologous recombination genes in Saccharomyces cerevisiae identifies multiple repair pathways for Sgs1, Mus81-Mms4 and RNase H2

Abstract

The DNA repair genes SGS1 and MUS81 of Saccharomyces cerevisiae are thought to control alternative pathways for the repair of toxic recombination intermediates based on the fact that sgs1Δ mus81Δ synthetic lethality is suppressed in the absence of homologous recombination (HR). Although these genes appear to functionally overlap in yeast and other model systems, the specific pathways controlled by SGS1 and MUS81 are poorly defined. Epistasis analyses based on DNA damage sensitivity previously indicated that SGS1 functioned primarily downstream of RAD51, and that MUS81 was independent of RAD51. To further define these genetic pathways, we carried out a systematic epistasis analysis between the RAD52-epistasis group genes and SGS1, MUS81, and RNH202, which encodes a subunit of RNase H2. Based on synthetic-fitness interactions and DNA damage sensitivities, we find that RAD52 is epistatic to MUS81 but not SGS1. In contrast, RAD54, RAD55 and RAD57 are epistatic to SGS1, MUS81 and RNH202. As expected, SHU2 is epistatic to SGS1, while both SHU1 and SHU2 are epistatic to MUS81. Importantly, loss of any RNase H2 subunit on its own resulted in increased recombination using a simple marker-excision assay. RNase H2 is thus needed to maintain genome stability consistent with the sgs1Δ rnh202Δ synthetic fitness defect. We conclude that SGS1 and MUS81 act in parallel pathways downstream of RAD51 and RAD52, respectively. The data further indicate these pathways share common components and display complex interactions.

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

“…These genetic interactions, as well as the lack of genetic interactions between an rnh203⌬ mutation and mutations in other genes, were confirmed by measuring the doubling times of different double mutants and their respective single mutants (Fig. 2 With the exception of the 8 new genetic interactions identified here, our results agreed well with previously reported mutations that do or do not interact with deletions of genes encoding RNase H2 (44,61,(64)(65)(66)(67)(68)(69)(70)(71). In addition, other studies have indicated that strains lacking RNH203 require DEP1, DNA2, MMS4 (encodes a subunit of the Mus81-Mms4 complex), RMI1 (encodes a subunit of the Sgs1-Rmi1-Top3 complex), RNH1, and SMC6 for normal growth (20,41,44,58,72), although we did not investigate these interactions in the present study.…”

“…In aggregate, the results from our analysis suggest that RNase H2 defects cause an accumulation of mutagenic damage in DNA that is then repaired or compensated for by different DNA metabolism pathways. The rad51⌬-mediated suppression of the slow-growth phenotype of specific rnh203⌬ double mutants indicates that the loss of RNase H2 activity in these mutants causes increased DNA damage during DNA replication, leading to the formation of aberrant DNA structures (44,55,65,75). Defects in HR, sister chromatid HR and end resection (rad52⌬, mre11⌬, rad50⌬, xrs2⌬, esc2⌬, smc6, and potentially, esc4⌬/rtt107⌬ and ctf4⌬) (85)(86)(87)(88), and resolution of branched HR intermediates (mus81⌬, top3⌬, sgs1⌬, mms4⌬, and mph1⌬) (61,69) all caused slow-growth interactions with RNase H2 defects.…”

“…Since ribonucleotides are more susceptible to spontaneous cleavage than deoxyribonucleotides (40), loss of RER or the presence of aberrant RER due to Top1 (30) could lead to increased strand breaks and, potentially, genome instability. In S. cerevisiae, RNase H2 defects cause weak mutator and hyperrecombination phenotypes (7,(41)(42)(43)(44)(45), and RNase H2 null mouse cells have increased chromosomal rearrangements and a p53-dependent DNA damage response (40,46). Interestingly, defects in RNase H2, as well as the Trex exonucleases, cause Aicardi-Goutieres syndrome, a neuroautoimmune disease that may result from inflammatory responses to aberrant DNA or RNA structures that accumulate as the result of these defects (10,47).…”

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

“…RNase H2-defective haploids, for example, have elevated gene conversion between closely linked repeats (Potenski et al 2014) and increased loss of markers flanked by direct repeats (Ii et al 2011). Although loss of either RNase H1 or RNase H2 promoted loss of an artificial chromosome, loss of both enzymes was required to elevate LOH on chromosome III in a diploid background (Wahba et al 2011).…”

“…In addition to the specific case of recombination associated with highly elevated transcription, yeast strains with reduced RNase H activity generally exhibit elevated genomic instability. Haploid strains lacking RNase H2, for example, have increased recombination between direct repeats (Ii et al 2011;Potenski et al 2014). Top1 is required for this increase in recombination, suggesting that the initiating lesion is a Top1-mediated nick at an rNMP (Potenski et al 2014).…”