We show by whole genome sequence analysis that loss of RNase H2 activity increases loss of heterozygosity (LOH) in Saccharomyces cerevisiae diploid strains harboring the pol2-M644G allele encoding a mutant version of DNA polymerase e that increases ribonucleotide incorporation. This led us to analyze the effects of loss of RNase H2 on LOH and on nonallelic homologous recombination (NAHR) in mutant diploid strains with deletions of genes encoding RNase H2 subunits (rnh201D, rnh202D, and rnh203D), topoisomerase 1 (TOP1D), and/or carrying mutant alleles of DNA polymerases e, a, and d. We observed an 7-fold elevation of the LOH rate in RNase H2 mutants encoding wild-type DNA polymerases. Strains carrying the pol2-M644G allele displayed a 7-fold elevation in the LOH rate, and synergistic 23-fold elevation in combination with rnh201D. In comparison, strains carrying the pol2-M644L mutation that decreases ribonucleotide incorporation displayed lower LOH rates. The LOH rate was not elevated in strains carrying the pol1-L868M or pol3-L612M alleles that result in increased incorporation of ribonucleotides during DNA synthesis by polymerases a and d, respectively. A similar trend was observed in an NAHR assay, albeit with smaller phenotypic differentials. The ribonucleotide-mediated increases in the LOH and NAHR rates were strongly dependent on TOP1. These data add to recent reports on the asymmetric mutagenicity of ribonucleotides caused by topoisomerase 1 processing of ribonucleotides incorporated during DNA replication. KEYWORDS LOH; NAHR; genome stability; recombination; ribonucleotides T HE replicative DNA polymerases of Saccharomyces cerevisiae, DNA polymerases a (Pol a), d (Pol d), and e (Pol e), frequently incorporate ribonucleotides into DNA both in vitro and during nuclear DNA replication in vivo (Nick McElhinny et al. 2010a,b;Williams and Kunkel 2014;Williams et al. 2015). These ribonucleotides are efficiently removed when RNase H2 incises the DNA backbone containing a ribonucleotide to initiate ribonucleotide excision repair (RER) (Nick McElhinny et al. 2010a;Sparks et al. 2012). When the RNH201 gene that encodes the catalytic subunit of RNase H2 (Cerritelli and Crouch 2009) is deleted, RER is defective and many unrepaired ribonucleotides remain in the genome. A subset of these unrepaired ribonucleotides can be removed when topoisomerase 1 (TOP1) incises a DNA backbone containing a ribonucleotide . However, TOP1 incision creates nicks with unligatable ends and elicits several RNA-DNA damage phenotypes, including slow growth, activation of the genome integrity checkpoint and altered progression through the cell cycle, sensitivity to the replication inhibitor hydroxyurea (HU), and strongly elevated rates for deletion of 2-5 bp from low-complexity DNA sequences (Nick McElhinny et al. 2010a;Clark et al. 2011;Kim et al. 2011). These effects are elicited primarily by ribonucleotides incorporated by Pol e, but not by ribonucleotides incorporated by Pol a or Pol d (Williams et al. 2015). Loss of RNase H2 is a...

The Saccharomyces cerevisiae strain JAY270/PE2 is a highly efficient biocatalyst used in the production of bioethanol from sugarcane feedstock. This strain is heterothallic and diploid, and its genome is characterized by abundant structural and nucleotide polymorphisms between homologous chromosomes. One of the reasons it is favored by many distilleries is that its cells do not normally aggregate, a trait that facilitates cell recycling during batch-fed fermentations. However, long-term propagation makes the yeast population vulnerable to the effects of genomic instability, which may trigger the appearance of undesirable phenotypes such as cellular aggregation. In pure cultures of JAY270, we identified the recurrent appearance of mutants displaying a mother-daughter cell separation defect resulting in rough colonies in agar media and fast sedimentation in liquid culture. We investigated the genetic basis of the colony morphology phenotype and found that JAY270 is heterozygous for a frameshift mutation in the ACE2 gene (ACE2/ace2-A7), which encodes a transcriptional regulator of mother-daughter cell separation. All spontaneous rough colony JAY270-derived isolates analyzed carried copy-neutral loss-of-heterozygosity (LOH) at the region of chromosome XII where ACE2 is located (ace2-A7/ace2-A7). We specifically measured LOH rates at the ACE2 locus, and at three additional chromosomal regions in JAY270 and in a conventional homozygous diploid laboratory strain. This direct comparison showed that LOH rates at all sites were quite similar between the two strain backgrounds. In this case study of genomic instability in an industrial strain, we showed that the JAY270 genome is dynamic and that structural changes to its chromosomes can lead to new phenotypes. However, our analysis also indicated that the inherent level of genomic instability in this industrial strain is normal relative to a laboratory strain. Our work provides an important frame of reference to contextualize the interpretation of instability processes observed in the complex genomes of industrial yeast strains.