Significance: Although geneticists often emphasize diversity generated during sexual reproduction, genomic alterations that occur during vegetative growth are an important source of genetic diversity. In the current study, we have measured the rate and location of single-base mutations, large deletions/duplications, ploidy alterations, and mitotic recombination events in extensively subcultured diploid isolates of the yeast Saccharomyces cerevisiae . We were able to determine the relative frequency of these events and to predict the frequency of loss of heterozygosity of allelic markers resulting from mitotic recombination. This analysis is relevant to understanding of genomic evolution, as well as the mitotic recombination events in humans that predispose cells to form tumors.

Oxidative DNA damage is a threat to genome stability. Using a genetic system in yeast that allows detection of mitotic recombination, we found that the frequency of crossovers is greatly elevated when cells are treated with hydrogen peroxide (H₂O₂). Using a combination of microarray analysis and genomic sequencing, we mapped the breakpoints of mitotic recombination events and other chromosome rearrangements at a resolution of about 1 kb. Gene conversions and crossovers were the two most common types of events, but we also observed deletions, duplications, and chromosome aneuploidy. In addition, H₂O₂-treated cells had elevated rates of point mutations (particularly A to T/T to A and C to G/G to C transversions) and small insertions/deletions (in/dels). In cells that underwent multiple rounds of H₂O₂ treatments, we identified a genetic alteration that resulted in improved H₂O₂ tolerance by amplification of the CTT1 gene that encodes cytosolic catalase T. Lastly, we showed that cells grown in the absence of oxygen have reduced levels of recombination. This study provided multiple novel insights into how oxidative stress affects genomic instability and phenotypic evolution in aerobic cells.

Although meiosis in warm-blooded organisms takes place in a narrow temperature range, meiosis in many organisms occurs over a wide variety of temperatures. We analyzed the properties of meiosis in the yeast Saccharomyces cerevisiae in cells sporulated at 14°C, 30°C, or 37°C. Using comparative-genomic-hybridization microarrays, we examined the distribution of Spo11-generated meiosis-specific double-stranded DNA breaks throughout the genome. Although there were between 300 and 400 regions of the genome with high levels of recombination (hot spots) observed at each temperature, only about 20% of these hot spots were found to have occurred independently of the temperature. In S. cerevisiae , regions near the telomeres and centromeres tend to have low levels of meiotic recombination. This tendency was observed in cells sporulated at 14°C and 30°C, but not at 37°C. Thus, the temperature of sporulation in yeast affects some global property of chromosome structure relevant to meiotic recombination. Using single-nucleotide polymorphism (SNP)-specific whole-genome microarrays, we also examined crossovers and their associated gene conversion events as well as gene conversion events that were unassociated with crossovers in all four spores of tetrads obtained by sporulation of diploids at 14°C, 30°C, or 37°C. Although tetrads from cells sporulated at 30°C had slightly (20%) more crossovers than those derived from cells sporulated at the other two temperatures, spore viability was good at all three temperatures. Thus, despite temperature-induced variation in the genetic maps, yeast cells produce viable haploid products at a wide variety of sporulation temperatures. IMPORTANCE In the yeast Saccharomyces cerevisiae , recombination is usually studied in cells that undergo meiosis at 25°C or 30°C. In a genome-wide analysis, we showed that the locations of genomic regions with high and low levels of meiotic recombination (hot spots and cold spots, respectively) differed dramatically in cells sporulated at 14°C, 30°C, and 37°C. Thus, in yeast, and likely in other non-warm-blooded organisms, genetic maps are strongly affected by the environment.

High temperature causes ubiquitous environmental stress to microorganisms, but studies have not fully explained whether and to what extent heat shock would affect genome stability. Hence, this study explored heat-shock-induced genomic alterations in the yeast Saccharomyces cerevisiae. Using genetic screening systems and customized single nucleotide polymorphism (SNP) microarrays, we found that heat shock (52 °C) for several minutes could heighten mitotic recombination by at least one order of magnitude. More than half of heat-shock-induced mitotic recombinations were likely to be initiated by DNA breaks in the S/G₂ phase of the cell cycle. Chromosomal aberration, mainly trisomy, was elevated hundreds of times in heat-shock-treated cells than in untreated cells. Distinct chromosomal instability patterns were also observed between heat-treated and carbendazim-treated yeast cells. Finally, we demonstrated that heat shock stimulates fast phenotypic evolutions (such as tolerance to ethanol, vanillin, fluconazole, and tunicamycin) in the yeast population. This study not only provided novel insights into the effect of temperature fluctuations on genomic integrity but also developed a simple protocol to generate an aneuploidy mutant of yeast.

Yeast strains with low levels of the replicative DNA polymerases (alpha, delta, and epsilon) have high levels of chromosome deletions, duplications, and translocations. By examining the patterns of mutations induced in strains with low levels of DNA polymerase by the human protein APOBEC3B (a protein that deaminates cytosine in single-stranded DNA), we show dramatically elevated amounts of single-stranded DNA relative to a wild-type strain. During DNA replication, one strand (defined as the leading strand) is replicated processively by DNA polymerase epsilon and the other (the lagging strand) is replicated as short fragments initiated by DNA polymerase alpha and extended by DNA polymerase delta. In the low DNA polymerase alpha and delta strains, the APOBEC-induced mutations are concentrated on the lagging-strand template, whereas in the low DNA polymerase epsilon strain, mutations occur on the leading- and lagging-strand templates with similar frequencies. In addition, for most genes, the transcribed strand is mutagenized more frequently than the nontranscribed strand. Lastly, some of the APOBEC-induced clusters in strains with low levels of DNA polymerase alpha or delta are greater than 10 kb in length. Significance Perturbations in DNA replication cause high levels of chromosome rearrangements and it has been suggested that DNA replication stress promotes oncogenesis. In this study, we show that low levels of the DNA polymerases involved in replication in the yeast Saccharomyces cerevisiae lead to greatly elevated levels of single-stranded DNA. We suggest that these single-stranded regions are fragile, generating the DNA breaks that initiate translocations and other types of chromosome rearrangements.

Baker’s yeast (Saccharomyces cerevisiae) is the common yeast used in the fields of bread making, brewing, and bioethanol production. Growth rate, stress tolerance, ethanol titer, and byproducts yields are some of the most important agronomic traits of S. cerevisiae for industrial applications. Here, we developed a novel method of constructing S. cerevisiae strains for co-producing bioethanol and ergosterol. The genome of an industrial S. cerevisiae strain, ZTW1, was first reconstructed through treatment with an antimitotic drug followed by sporulation and hybridization. A total of 140 mutants were selected for ethanol fermentation testing, and a significant positive correlation between ergosterol content and ethanol production was observed. The highest performing mutant, ZG27, produced 7.9 % more ethanol and 43.2 % more ergosterol than ZTW1 at the end of fermentation. Chromosomal karyotyping and proteome analysis of ZG27 and ZTW1 suggested that this breeding strategy caused large-scale genome structural variations and global gene expression diversities in the mutants. Genetic manipulation further demonstrated that the altered expression activity of some genes (such as ERG1, ERG9, and ERG11) involved in ergosterol synthesis partly explained the trait improvement in ZG27.