Ionizing radiation is an established source of chromosome aberrations (CAs). Although double-strand breaks (DSBs) are implicated in radiation-induced and other CAs, the underlying mechanisms are poorly understood. Here, we show that, although the vast majority of randomly induced DSBs in G ₂ diploid yeast cells are repaired efficiently through homologous recombination (HR) between sister chromatids or homologous chromosomes, ≈2% of all DSBs give rise to CAs. Complete molecular analysis of the genome revealed that nearly all of the CAs resulted from HR between nonallelic repetitive elements, primarily Ty retrotransposons. Nonhomologous end-joining (NHEJ) accounted for few, if any, of the CAs. We conclude that only those DSBs that fall at the 3–5% of the genome composed of repetitive DNA elements are efficient at generating rearrangements with dispersed small repeats across the genome, whereas DSBs in unique sequences are confined to recombinational repair between the large regions of homology contained in sister chromatids or homologous chromosomes. Because repeat-associated DSBs can efficiently lead to CAs and reshape the genome, they could be a rich source of evolutionary change.

Significance: Chromosomes of most organisms have regions of high GC content interspersed with regions of low GC content. We constructed three variants of the yeast URA3 gene with GC contents of 31%, 43%, and 63%. We found that the high-GC URA3 gene had a substantially elevated rate of mutations, both single-base substitutions and deletions. The elevated base substitutions require an error-prone DNA polymerase, and the high rate of deletions occurs as a consequence of DNA polymerase slippage. The high-GC gene also had substantially elevated rates of mitotic and meiotic recombination. These observations indicate that GC content is an important parameter influencing genome evolution.

Homologous recombination is an important mechanism for the repair of DNA damage in mitotically dividing cells. Mitotic crossovers between homologues with heterozygous alleles can produce two homozygous daughter cells (loss of heterozygosity), whereas crossovers between repeated genes on non-homologous chromosomes can result in translocations. Using a genetic system that allows selection of daughter cells that contain the reciprocal products of mitotic crossing over, we mapped crossovers and gene conversion events at a resolution of about 4 kb in a 120-kb region of chromosome V of Saccharomyces cerevisiae. The gene conversion tracts associated with mitotic crossovers are much longer (averaging about 12 kb) than the conversion tracts associated with meiotic recombination and are non-randomly distributed along the chromosome. In addition, about 40% of the conversion events have patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle. Most higher organisms have two copies of several different types of chromosomes. For example, the human female has 23 pairs of chromosomes. Although the chromosome pairs have very similar sequences, they are not identical. Members of a chromosome pair can swap segments from one chromosome to the other; these exchanges are called “recombination.” Most previous studies of recombination have been done in cells undergoing meiosis, the process that leads to the formation of eggs and sperm (gametes). Recombination, however, can also occur in cells that are dividing mitotically. In our study, we examine the properties of mitotic recombination in yeast. We show that mitotic recombination differs from meiotic recombination in two important ways. First, the sizes of the chromosome segments that are non-reciprocally transferred during mitotic recombination are much larger than those transferred during meiotic exchange. Second, in meiosis, most recombination events involve the repair of a single chromosome break, whereas in mitosis, about half of the recombination events appear to involve the repair of two chromosome breaks.

Although homologous recombination is an important pathway for the repair of double-stranded DNA breaks in mitotically dividing eukaryotic cells, these events can also have negative consequences, such as loss of heterozygosity (LOH) of deleterious mutations. We mapped about 140 spontaneous reciprocal crossovers on the right arm of the yeast chromosome IV using single-nucleotide-polymorphism (SNP) microarrays. Our mapping and subsequent experiments demonstrate that inverted repeats of Ty retrotransposable elements are mitotic recombination hotspots. We found that the mitotic recombination maps on the two homologs were substantially different and were unrelated to meiotic recombination maps. Additionally, about 70% of the DNA lesions that result in LOH are likely generated during G1 of the cell cycle and repaired during S or G2. We also show that different genetic elements are associated with reciprocal crossover conversion tracts depending on the cell cycle timing of the initiating DSB. Double-strand breaks (DSBs) are DNA lesions that can be fatal to a cell if left unrepaired. They can be caused by exogenous sources, such as gamma radiation, or endogenous stresses, such as high levels of transcription. Yeast cells primarily repair DSBs that are initiated outside of meiosis by mitotic recombination, which can result in physical exchanges between chromosomes, known as crossovers. We created a mitotic recombination map of one chromosome arm, representing 10% of the genome. This recombination map allows us to determine which regions of the chromosome arm are more susceptible to DNA damage than other regions. We were able to determine that most DSBs that result in detectable genomic changes were initiated prior to DNA replication and that some secondary DNA structures can be recombination hotspots. Recombination can also occur during meiosis, as a method of ensuring proper chromosome segregation. However, previously reported meiotic recombination maps have no correlation with our mitotic recombination map.

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.

Significance: When mammalian cells are treated with drugs that perturb DNA replication, chromosomes break in specific locations called “fragile sites.” Human fragile sites are hotspots for chromosome rearrangements seen in many solid tumors. In the yeast Saccharomyces cerevisiae , DNA breaks are repaired by homologous recombination. We have developed methods of genome-wide mapping of recombination events in yeast strains that have 10-fold reduced levels of DNA polymerase. We find that these yeast fragile sites are often associated with sequence/structural motifs that pause or stall the DNA replication fork. We also show that low levels of DNA polymerase greatly elevate the frequency of deletions and duplications (reflecting unequal sister-chromatid recombination between repeated genes), and result in changes in chromosome number (aneuploidy).