In the yeast Saccharomyces cerevisiae, meiotic recombination is initiated by double-strand DNA breaks (DSBs). Meiotic DSBs occur at relatively high frequencies in some genomic regions (hotspots) and relatively low frequencies in others (coldspots). We used DNA microarrays to estimate variation in the level of nearby meiotic DSBs for all 6,200 yeast genes. Hotspots were nonrandomly associated with regions of high G ؉ C base composition and certain transcriptional profiles. Coldspots were nonrandomly associated with the centromeres and telomeres. Hotspots are genomic regions with unusually high levels of recombination (1). From studies in yeast, several generalizations concerning hotspots can be made. First, most hotspots are intergenic rather than intragenic (2). Second, genetically defined hotspots are associated with local double-strand DNA breaks (DSBs) (1). Third, DSBs usually occur in DNase Isensitive regions (3). Fourth, activity of the HIS4 hotspot in Saccharomyces cerevisiae requires the binding of transcription factors in the hotspot region (4-6), but does not require high levels of meiosis-specific transcription (7). Hotspots that require transcription factor binding are called ␣ hotspots (8). Fifth, certain DNA sequences result in hotspots (␤ hotspots) that do not require the binding of known transcription factors (9).Meiotic DSB formation requires Spo11p, a topoisomerase II-related protein that is transiently covalently attached to the 5Ј ends of the DNA fragments (10, 11). In wild-type yeast cells, the Spo11p is removed to allow subsequent steps in recombination. In strains with the rad50S mutation, however, Spo11p stays covalently attached to the broken DNA ends (11).Coldspots in yeast have received less attention than hotspots. Lambie and Roeder (12) showed that the centromere of chromosome III reduced crossing-over and gene conversion of nearby markers, and Baudat and Nicolas (13) noted a lack of DSB sites near the centromere. Several researchers have found a relative lack of DSB sites in rad50S strains near the telomeres (13,14).Although observations concerning individual hotspots and coldspots have given clues as to the mechanism of recombination initiation, our ability to predict hotspots and coldspots from DNA sequence information is very limited. A complementary approach is to map hotspots and coldspots globally and to determine whether they share common DNA sequences and͞or structural elements. Such mapping studies have been performed to map DSB sites to single-gene resolution on chromosome III (13) and to the resolution of a pulsed-field gel on chromosomes I, III, and VI (14,15). By using DNA samples enriched for meiosis-specific DSBs as hybridization probes on DNA microarrays, we extended these analyses to measure the global distribution of DSBs at single-gene resolution. Materials and MethodsYeast Strains. FX4, FX6, and QFY105 are diploid rad50S strains that have been described (4). These strains (used in the microarray analysis) are isogenic except for changes introduced by transformati...

Meiotic recombination events are distributed unevenly throughout eukaryotic genomes. This inhomogeneity leads to distortions of genetic maps that can hinder the ability of geneticists to identify genes by map-based techniques. Various lines of evidence, particularly from studies of yeast, indicate that the distribution of recombination events might reflect, at least in part, global features of chromosome structure, such as the distribution of modified nucleosomes.

In the yeast Saccharomyces cerevisiae, reduced levels of the replicative alpha DNA polymerase result in greatly elevated frequencies of chromosome translocations and chromosome loss. We selected translocations in a small region of chromosome III and found that they involve homologous recombination events between yeast retrotransposons (Ty elements) on chromosome III and retrotransposons located on other chromosomes. One of the two preferred sites of these translocations on chromosome III involve two Ty elements arrayed head-to-head; disruption of this site substantially reduces the rate of translocations. We demonstrate that this pair of Ty elements constitutes a preferred site for double-strand DNA breaks when DNA replication is compromised, analogous to the fragile sites observed in mammalian chromosomes.

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...

Yeast chromosomes terminate in tracts of simple repetitive DNA (poly[G1-3T]). Mutations in the gene TEL1 result in shortened telomeres. Sequence analysis of TEL1 indicates that it encodes a very large (322 kDa) protein with amino acid motifs found in phosphatidylinositol/protein kinases. The closest homolog to TEL1 is the human ataxia telangiectasia gene.