Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the rates of all of these genetic changes in a diploid strain of Saccharomyces cerevisiae by whole-genome sequencing of many independent isolates (n = 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n = 1,337) and mitotic recombination events (n = 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.

DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G 2 phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the lowpolymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/ deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.DNA replication stress | DNA polymerase | genome instability I n normal rapidly dividing cells, DNA replication is rapid and accurate, preventing the accumulation of genomic alterations. Stalling of replication forks or inappropriate initiation of replication origins can result in DNA replication stress (DRS) that can contribute to cancer development (1). It has been proposed that mutations in oncogenes and tumor suppressor genes drive cell proliferation and induce DRS. In turn, DRS generates genome instability, allowing cells with various types of genetic variations (mutations, duplications, translocations) to escape cellular senescence and apoptosis (2). However, the mechanisms by which oncogenes induce DRS and the precise nature of DRSassociated DNA lesions have not been clearly defined.Exposure of mammalian cells in culture to conditions that perturb DNA synthesis result in "fragile sites," gaps or constrictions detected by light microscopy in metaphase chromosomes (3). Aphidicolin, a drug that inhibits DNA polymerase, is one agent that induces fragile sites. The break points of chromosome rearrangements that occur in tumor cells often colocalize with fragile sites (3, 4), establishing another link between DRS and cancer. In addition to inducing chromosome breaks in cultured cells, aphidicolin induces high frequencies of duplications and deletions similar to those observed in tumor cells (5).As a model for mammalian fragile sites, we previously constructed yeast strains in which the transcription of the replicative DNA polymerases α (encoded by POL1) or δ (encoded by POL3) was regulated by the GAL1 promoter (6-8). Under lowgalactose growth conditions, which reduced the levels of DNA polymerases α or δ about 10-fold, these strains had elevated rates of chromosome loss and rearrangements on chromosome III.We also studied g...