We examined the stability of microsatellites of different repeat unit lengths in Saccharomyces cerevisiae strains deficient in DNA mismatch repair. The msh2 and msh3 mutations destabilized microsatellites with repeat units of 1, 2, 4, 5, and 8 bp; a poly(G) tract of 18 bp was destabilized several thousand-fold by the msh2 mutation and about 100-fold by msh3. The msh6 mutations destabilized microsatellites with repeat units of 1 and 2 bp but had no effect on microsatellites with larger repeats. These results argue that coding sequences containing repetitive DNA tracts will be preferred target sites for mutations in human tumors with mismatch repair defects. We find that the DNA mismatch repair genes destabilize microsatellites with repeat units from 1 to 13 bp but have no effect on the stability of minisatellites with repeat units of 16 or 20 bp. Our data also suggest that displaced loops on the nascent strand, resulting from DNA polymerase slippage, are repaired differently than loops on the template strand.Eukaryotic genomes often contain regions of DNA (called microsatellites or minisatellites) in which a single base or a small number of bases is tandemly repeated. In this paper, repetitive tracts with repeats of 1 to 13 bp will be considered microsatellites and tracts with repeats of more than 16 bp will be considered minisatellites. Both microsatellites and minisatellites are unstable, frequently undergoing deletions and additions (10,13,19). In vitro replication experiments demonstrate that DNA polymerase frameshift errors occur in repetitive sequences (17), and most of the available in vivo data suggest that alterations in microsatellite length reflect DNA polymerase slippage events (19,27). This mechanism predicts a transient dissociation of the template and the nascent strand during replication of the microsatellite (28). Due to the repetitive nature of the tract, the two DNA strands can reassociate out of register, leaving one or more unpaired repeats on either the template or nascent strand (see Fig. 1). If the distortion caused by these unpaired bases is not removed from the newly synthesized strand by the DNA mismatch repair system, the result will be a loss (if the unpaired bases are on the template strand) or a gain (if the unpaired bases are on the nascent strand) of one or more repeats. As expected from this model, mutations in the genes required for DNA mismatch repair greatly increase the rate of instability of repetitive DNA sequences in Escherichia coli, the yeast Saccharomyces cerevisiae, and human cells (19,22,27).In E. coli, two of the proteins involved in DNA mismatch repair are MutS (involved in recognition of the DNA mismatch) and MutL (involved in interactions between MutS and other proteins) (22). Homologs of these proteins have been identified in yeast and mammals. In yeast, the effects of mutations in the mutL homologs MLH1 and PMS1 and in the mutS homologs MSH2, MSH3, and MSH6 on the stability of a 33-bp poly(GT) repeat have been examined previously (14,26,27). Mutations in MLH1, P...

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (;2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

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.

In the yeast Saccharomyces cerevisiae, certain genomic regions have very high levels of meiotic recombination (hot spots). The hot spot activity associated with the HIS4 gene requires the Bas1p transcription factor. To determine whether this relationship between transcription factor binding and hot spot activity is general, we used DNA microarrays to map all genomic Bas1p binding sites and to map the frequency of meiosis-specific double-strand DNA breaks (as an estimate of the recombination activity) of all genes in both wild-type and bas1 strains. We identified sites of Bas1p-DNA interactions upstream of 71 genes, many of which are involved in histidine and purine biosynthesis. Our analysis of recombination activity in wild-type and bas1 strains showed that the recombination activities of some genes with Bas1p binding sites were dependent on Bas1p (as observed for HIS4), whereas the activities of other genes with Bas1p binding sites were unaffected or were repressed by Bas1p. These data demonstrate that the effect of transcription factors on meiotic recombination activity is strongly context dependent. In wild-type and bas1 strains, meiotic recombination was strongly suppressed in large (25-to 150-kb) chromosomal regions near the telomeres and centromeres and in the region flanking the rRNA genes. These results argue that both local and regional factors affect the level of meiotic recombination.From comparisons of genetic and physical maps, it is clear that recombination events are unevenly distributed. Regions with relatively high and low levels of exchange are termed "hot spots" and "cold spots," respectively. As first shown for Saccharomyces cerevisiae (28), meiotic recombination events in many eukaryotes (including humans) are initiated by doublestrand DNA breaks (DSBs) catalyzed by Spo11p, a topoisomerase II-related protein. In general, there is a good correlation between the frequency of DSBs and the rate of local meiotic recombination (36, 43). In the study described here, we use DNA microarrays to measure the rate of DSBs for all open reading frames (ORFs) and intergenic regions. We assume that these measurements will reflect the meiotic recombination activities near the DSB sites, although the nature of the later steps of recombination (strand invasion, extent of heteroduplex formation, etc.) could influence the recombination frequency.From studies of individual hot spots in Saccharomyces cerevisiae, as well as from more global studies, several generalizations concerning hot spots and cold spots can be made. First, DSBs usually occur in intergenic regions rather than within genes (4, 22, 58), suggesting a connection between "open" chromatin and preferred sites for Spo11p-induced cleavage. Second, for some hot spots (␣ hot spots), binding of transcription factors is required for hot spot activity (56) and DSB formation (19). This requirement for transcription factor binding does not indicate a direct connection between transcription and hot spot activity, since deletion of a TATAA sequence, which substa...