Margaret Dominska

Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes.

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.

Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production

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 (approximately 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.

Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences.

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

Genetic regulation of telomere-telomere fusions in the yeast Saccharomyces cerevisae

Yeast strains with mutations in both TEL1 and MEC1 have short telomeres and elevated rates of chromosome deletions. By using a PCR assay, we demonstrate that mec1 tel1 strains also have telomere–telomere fusions (T-TFs). T-TFs require Lig4p (a ligase required for nonhomologous end-joining DNA repair). The highest rates of T-TFs are found in strains with combination of mutations that affect telomere length and DNA damage checkpoints (mec1 tel1, mec3 tel1, mre11 mec1, and ddc1 tel1 strains). Examining many mutant genotypes, we find good agreement between the level of T-TFs and the rate of chromosomal deletions. In addition, if telomeres are elongated in a mec1 tel1 strain, we eliminate T-TFs and reduce the deletion rate. The correlation between the level of T-TFs and the rate of deletions argues that many of these deletions reflect a cycle of T-TF formation (resulting in dicentric chromosomes), followed by chromosome breakage.

A Fine-Structure Map of Spontaneous Mitotic Crossovers in the Yeast Saccharomyces cerevisiae

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.

Control of meiotic recombination and gene expression in yeast by a simple repetitive DNA sequence that excludes nucleosomes.

ABSTRACT Tandem repeats of the pentanucleotide 5′-CCGNN (where N indicates any base) were previously shown to exclude nucleosomes in vitro (Y.-H. Wang and J. D. Griffith, Proc. Natl. Acad. Sci. USA 93:8863–8867, 1996). To determine the in vivo effects of these sequences, we replaced the upstream regulatory sequences of the HIS4 gene ofSaccharomyces cerevisiae with either 12 or 48 tandem copies of CCGNN. Both tracts activated HIS4 transcription. We found that (CCGNN)12 tracts elevated meiotic recombination (hot spot activity), whereas the (CCGNN)48 tract repressed recombination (cold spot activity). In addition, a “pure” tract of (CCGAT)12 activated both transcription and meiotic recombination. We suggest that the cold spot activity of the (CCGNN)48 tract is related to the phenomenon of the suppressive interactions of adjacent hot spots previously described in yeast (Q.-Q. Fan, F. Xu, and T. D. Petes, Mol. Cell. Biol. 15:1679–1688, 1995; Q.-Q. Fan, F. Xu, M. A. White, and T. D. Petes, Genetics 145:661–670, 1997; T.-C. Wu and M. Lichten, Genetics 140:55–66, 1995; L. Xu and N. Kleckner, EMBO J. 16:5115–5128, 1995).

Loss of a histone deacetylase dramatically alters the genomic distribution of Spo11p-catalyzed DNA breaks in Saccharomyces cerevisiae

In eukaryotes, meiotic recombination events are distributed nonrandomly in the genome, with certain regions having high levels of recombination (hotspots) and others having low levels (coldspots). Species with similar DNA sequences (for example, chimpanzees and humans) can have strikingly different patterns of hotspots and coldspots. Below, by using a microarray analysis that allows us to measure the frequency of the meiosis-specific double-strand DNA breaks (DSBs) of all 6,000 yeast genes, we show that mutation of a single gene (SIR2), which encodes a histone deacetylase, significantly changes DSB frequencies of 12% of yeast genes, elevating DSBs of 5%, and reducing DSBs of 7%. Many of the genes with repressed recombination are located in large (50–100 kb) regions located near, but not at, the telomeres. Some of the genes with altered frequencies of DSBs (including the ribosomal RNA gene cluster) are known targets of Sir2p deacetylation in the wild-type strain.

Global Analysis of the Relationship between the Binding of the Bas1p Transcription Factor and Meiosis-Specific Double-Strand DNA Breaks in Saccharomyces cerevisiae†

ABSTRACT 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.

The histone methylase Set2p and the histone deacetylase Rpd3p repress meiotic recombination at the HIS4 meiotic recombination hotspot in Saccharomyces cerevisiae.

The rate of meiotic recombination in the yeast Saccharomyces cerevisiae varies widely in different regions of the genome with some genes having very high levels of recombination (hotspots). A variety of experiments done in yeast suggest that hotspots are a feature of chromatin structure rather than a feature of primary DNA sequence. We examined the effects of mutating a variety of enzymes that affect chromatin structure on the recombination activity of the well-characterized HIS4 hotspot including the Set2p and Dot1p histone methylases, the Hda1p and Rpd3p histone deacetylases, the Sin4p global transcription regulator, and a deletion of one of the two copies of the genes encoding histone H3-H4. Loss of Set2p or Rpd3p substantially elevated HIS4 hotspot activity, and loss of Hda1p had a smaller stimulatory effect; none of the other alterations had a significant effect. The increase of HIS4 hotspot activity in set2 and rpd3 strains is likely to be related to the recent finding that histone H3 methylation by Set2p directs deacetylation of histones by Rpd3p.

Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae

In eukaryotes, mutations in a number of genes that affect DNA damage checkpoints or DNA replication also affect telomere length [Curr. Opin. Cell Biol. 13 (2001) 281]. Saccharomyces cerevisae strains with mutations in the TEL1 gene (encoding an ATM-like protein kinase) have very short telomeres, as do strains with mutations in XRS2, RAD50, or MRE11 (encoding members of a trimeric complex). Xrs2p and Mre11p are phosphorylated in a Tel1p-dependent manner in response to DNA damage [Genes Dev. 15 (2001) 2238; Mol. Cell 7 (2001) 1255]. We found that Xrs2p, but not Mre11p or Rad50p, is efficiently phosphorylated in vitro by immunopreciptated Tel1p. Strains with mutations eliminating all SQ and TQ motifs in Xrs2p (preferred targets of the ATM kinase family) had wild-type length telomeres and wild-type sensitivity to DNA damaging agents. We also showed that Rfa2p (a subunit of RPA) and the Dun1p checkpoint kinase, which are required for DNA damage repair and which are phosphorylated in response to DNA damage in vivo, are in vitro substrates of the Tel1p and Mec1p kinases. In addition, Dun1p substrates with no SQ or TQ motifs are phosphorylated by Mec1p in vitro very inefficiently, but retain most of their ability to be phosphorylated by Tel1p. We demonstrated that null alleles of DUN1 and certain mutant alleles of RFA2 result in short telomeres. As observed with Xrs2p, however, strains with mutations of DUN1 or RFA2 that eliminate SQ motifs have no effect on telomere length or DNA damage sensitivity.