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...

While gene copy number variations (CNVs) are abundant in the human genome, and often are associated with disease consequences, the mutagenic pathways and environmental exposures that cause these large structural mutations are understudied relative to conventional nucleotide substitutions in DNA. The members of the environmental mutagenesis community are currently seeking to remedy this deficiency, and there is a renewed interest in the development of mutagenicity assays to identify and characterize compounds that may induce de novo CNVs in humans. To achieve this goal, it is critically important to acknowledge that CNVs exist in two very distinct classes: nonrecurrent and recurrent CNVs. The goal of this commentary is to emphasize the deep contrasts that exist between the proposed pathways that lead to these two mutation classes. Nonrecurrent de novo CNVs originate primarily in mitotic cells through replication-dependent DNA repair pathways that involve microhomologies (<10 bp), and are detected at higher frequency in children of older fathers. In contrast, recurrent de novo CNVs are most often formed in meiotic cells through homologous recombination between nonallelic large low-copy repeats (>10,000 bp), without an associated paternal age effect. Given the biological differences between the two CNV classes, it is our belief that nonrecurrent and recurrent CN mutagens will probably differ substantially in their modes of action. Therefore, each CNV class may require their own uniquely designed assays, so that we as a field may succeed in uncovering the broadest possible spectrum of environmental CN mutagens.