Isolation and characterization of point mutations in mismatch repair genes that destabilize microsatellites in yeast.

The stability of simple repetitive DNA sequences (microsatellites) is a sensitive indicator of the ability of a cell to repair DNA mismatches. In a genetic screen for yeast mutants with elevated microsatellite instability, we identified strains containing point mutations in the yeast mismatch repair genes, MSH2, MSH3, MLH1, and PMS1. Some of these mutations conferred phenotypes significantly different from those of null mutations in these genes. One semidominant MSH2 mutation was identified. Finally we showed that strains heterozygous for null mutations of mismatch repair genes in diploid strains in yeast confer subtle defects in the repair of small DNA loops.

Meiotic recombination involving heterozygous large insertions in Saccharomyces cerevisiae: formation and repair of large, unpaired DNA loops.

Meiotic recombination in Saccharomyces cerevisiae involves the formation of heteroduplexes, duplexes containing DNA strands derived from two different homologues. If the two strands of DNA differ by an insertion or deletion, the heteroduplex will contain an unpaired DNA loop. We found that unpaired loops as large as 5.6 kb can be accommodated within a heteroduplex. Repair of these loops involved the nucleotide excision repair (NER) enzymes Rad1p and Rad10p and the mismatch repair (MMR) proteins Msh2p and Msh3p, but not several other NER (Rad2p and Rad14p) and MMR (Msh4p, Msh6p, Mlh1p, Pms1p, Mlh2p, Mlh3p) proteins. Heteroduplexes were also formed with DNA strands derived from alleles containing two different large insertions, creating a large "bubble"; repair of this substrate was dependent on Rad1p. Although meiotic recombination events in yeast are initiated by double-strand DNA breaks (DSBs), we showed that DSBs occurring within heterozygous insertions do not stimulate interhomologue recombination.

Meiotic recombination hot spots and cold spots.

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.

The Saccharomyces cerevisiae suppressor of choline sensitivity (SCS2) gene is a multicopy Suppressor of mec1 telomeric silencing defects.

Mec1p is a cell cycle checkpoint protein related to the ATM protein kinase family. Certain mec1 mutations or overexpression of Mec1p lead to shortened telomeres and loss of telomeric silencing. We conducted a multicopy suppressor screen for genes that suppress the loss of silencing in strains overexpressing Mec1p. We identified SCS2 (suppressor of choline sensitivity), a gene previously isolated as a suppressor of defects in inositol synthesis. Deletion of SCS2 resulted in decreased telomeric silencing, and the scs2 mutation increased the rate of cellular senescence observed for mec1-21 tel1 double mutant cells. Genetic analysis revealed that Scs2p probably acts through a different telomeric silencing pathway from that affected by Mec1p.

Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations.

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.

Decreased meiotic intergenic recombination and increased meiosis I nondisjunction in exo1 mutants of Saccharomyces cerevisiae.

Exonuclease I was originally identified as a 5' --> 3' deoxyribonuclease present in fractionated extracts of Schizosaccharomyces pombe and Saccharomyces cerevisiae. Genetic analysis of exo1 mutants of both yeasts revealed no major defect in meiosis, suggesting that exonuclease I is unlikely to be the primary activity that processes meiosis-specific double-strand breaks (DSBs). We report here that exo1 mutants of S. cerevisiae exhibit subtle but complex defects in meiosis. Diploids containing a homozygous deletion of EXO1 show decreased spore viability associated with an increase in meiosis I nondisjunction, while intergenic recombination is reduced about twofold. Exo1p functions in the same pathway as Msh5p for intergenic recombination. The length of heteroduplex tracts within the HIS4 gene is unaffected by the exo1 mutation. These results suggest that Exo1p is unlikely to play a major role in processing DSBs to form single-stranded tails at HIS4, but instead appears to promote crossing over to ensure disjunction of homologous chromosomes. In addition, our data indicate that exonuclease I may have a minor role in the correction of large DNA mismatches that occur in heteroduplex DNA during meiotic recombination at the HIS4 locus.

Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase.

The Saccharomyces cerevisiae proteins Tel1p and Mec1p are involved in telomere length regulation and cellular responses to DNA damage. The closest relative of these proteins is the human Ataxia Telangiectasia Mutated (ATM) protein, a wortmannin-sensitive protein kinase that primarily phosphorylates serines in an SQ motif. We constructed yeast strains containing functional epitope-tagged versions of Tel1p and Mec1p. We showed that immunoprecipitated Tel1p and Mec1p were capable of in vitro phosphorylation of the mammalian protein PHAS-I (Phosphorylated Heat and Acid Stable protein). These activities are sensitive to wortmannin. Tel1p phosphorylates serine in an SQ motif in PHAS-I. Mutations in the kinase domains of Tel1p and Mec1p result in loss of in vitro kinase activity and the in vivo phenotypes associated with the null tel1 and mec1 mutations.

Global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae.

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.

Increased rates of genomic deletions generated by mutations in the yeast gene encoding DNA polymerase delta or by decreases in the cellular levels of DNA polymerase delta.

In Saccharomyces cerevisiae, POL3 encodes the catalytic subunit of DNA polymerase delta. While yeast POL3 mutant strains that lack the proofreading exonuclease activity of the polymerase have a strong mutator phenotype, little is known regarding the role of other Pol3p domains in mutation avoidance. We identified a number of pol3 mutations in regions outside of the exonuclease domain that have a mutator phenotype, substantially elevating the frequency of deletions. These deletions appear to reflect an increased frequency of DNA polymerase slippage. In addition, we demonstrate that reduction in the level of wild-type DNA polymerase results in a similar mutator phenotype. Lowered levels of DNA polymerase also result in increased sensitivity to the DNA-damaging agent methyl methane sulfonate. We conclude that both the quantity and the quality of DNA polymerase delta is important in ensuring genome stability.

The Mre11p/Rad50p/Xrs2p complex and the Tel1p function in a single pathway for telomere maintenance in yeast.

The Mre11p/Rad50p/Xrs2p complex is involved in the repair of double-strand DNA breaks, nonhomologous end joining, and telomere length regulation. TEL1 is primarily involved in telomere length regulation. By an epistasis analysis, we conclude that Tel1p and the Mre11p/Rad50p/Xrs2p complex function in a single pathway of telomere length regulation.

Involvement of the checkpoint protein Mec1p in silencing of gene expression at telomeres in Saccharomyces cerevisiae.

Yeast strains with a mutation in the MEC1 gene are deficient in the cellular checkpoint response to DNA-damaging agents and have short telomeres (K. B. Ritchie, J. C. Mallory, and T. D. Petes, Mol. Cell. Biol. 19:6065-6075, 1999; T. A. Weinert, G. L. Kiser, and L. H. Hartwell, Genes Dev. 8:652-665, 1994). In wild-type yeast cells, genes inserted near the telomeres are transcriptionally silenced (D. E. Gottschling, O. M. Aparichio, B. L. Billington, and V. A. Zakian, Cell 63:751-762, 1990). We show that mec1 strains have reduced ability to silence gene expression near the telomere. This deficiency was alleviated by the sml1 mutation. Overexpression of Mec1p also resulted in a silencing defect, although this overexpression did not affect the checkpoint function of Mec1p. Telomeric silencing was not affected by mutations in several other genes in the Mec1p checkpoint pathway (null mutations in RAD9 and CHK1 or in several hypomorphic rad53 alleles) but was reduced by a null mutation of DUN1. In addition, the loss of telomeric silencing in mec1 strains was not a consequence of the slightly shortened telomeres observed in these strains.

Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae.

In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (arg8(m)) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome.

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

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 of Saccharomyces 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).

Interactions of TLC1 (which encodes the RNA subunit of telomerase), TEL1, and MEC1 in regulating telomere length in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, chromosomes terminate with a repetitive sequence [poly(TG(1-3))] 350 to 500 bp in length. Strains with a mutation of TEL1, a homolog of the human gene (ATM) mutated in patients with ataxia telangiectasia, have short but stable telomeric repeats. Mutations of TLC1 (encoding the RNA subunit of telomerase) result in strains that have continually shortening telomeres and a gradual loss of cell viability; survivors of senescence arise as a consequence of a Rad52p-dependent recombination events that amplify telomeric and subtelomeric repeats. We show that a mutation in MEC1 (a gene related in sequence to TEL1 and ATM) reduces telomere length and that tel1 mec1 double mutant strains have a senescent phenotype similar to that found in tlc1 strains. As observed in tlc1 strains, survivors of senescence in the tel1 mec1 strains occur by a Rad52p-dependent amplification of telomeric and subtelomeric repeats. In addition, we find that strains with both tel1 and tlc1 mutations have a delayed loss of cell viability compared to strains with the single tlc1 mutation. This result argues that the role of Tel1p in telomere maintenance is not solely a direct activation of telomerase.

Dependence of the regulation of telomere length on the type of subtelomeric repeat in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, chromosomes terminate with approximately 400 bp of a simple repeat poly(TG(1-3)). Based on the arrangement of subtelomeric X and Y' repeats, two types of yeast telomeres exist, those with both X and Y' (Y' telomeres) and those with only X (X telomeres). Mutations that result in abnormally short or abnormally long poly(TG(1-3)) tracts have been previously identified. In this study, we investigated telomere length in strains with two classes of mutations, one that resulted in short poly(TG(1-3)) tracts (tel1) and one that resulted in elongated tracts (pif1, rap1-17, rif1, or rif2). In the tel1 pif1 strain, Y' telomeres had about the same length as those in tel1 strains and X telomeres had lengths intermediate between those in tel1 and pif1 strains. Strains with either the tel1 rap1-17 or tel1 rif2 genotypes had short tracts for all chromosome ends examined, demonstrating that the telomere elongation characteristic of rap1-17 and rif2 strains is Tel1p-dependent. In strains of the tel1 rif1 or tel1 rif1 rif2 genotypes, telomeres with Y' repeats had short terminal tracts, whereas most of the X telomeres had long terminal tracts. These results demonstrate that the regulation of telomere length is different for X and Y' telomeres.

Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent).

Triplet repeats form secondary structures that escape DNA repair in yeast.

Several human neurodegenerative diseases result from expansion of CTG/CAG or CGG/CCG triplet repeats. The finding that single-stranded CNG repeats form hairpin-like structures in vitro has led to the hypothesis that DNA secondary structure formation is an important component of the expansion mechanism. We show that single-stranded DNA loops containing 10 CTG/CAG or CGG/CCG repeats are inefficiently repaired during meiotic recombination in Saccharomyces cerevisiae. Comparisons of the repair of DNA loops with palindromic and nonpalindromic sequences suggest that this inefficient repair reflects the ability of these sequences to form hairpin structures in vivo.

A mutation of the yeast gene encoding PCNA destabilizes both microsatellite and minisatellite DNA sequences.

The POL30 gene of the yeast Saccharomyces cerevisiae encodes the proliferating cell nuclear antigen (PCNA), a protein required for processive DNA synthesis by DNA polymerase delta and epsilon. We examined the effects of the pol30-52 mutation on the stability of microsatellite (1- to 8-bp repeat units) and minisatellite (20-bp repeat units) DNA sequences. It had previously been shown that this mutation destabilizes dinucleotide repeats 150-fold and that this effect is primarily due to defects in DNA mismatch repair. From our analysis of the effects of pol30-52 on classes of repetitive DNA with longer repeat unit lengths, we conclude that this mutation may also elevate the rate of DNA polymerase slippage. The effect of pol30-52 on tracts of repetitive DNA with large repeat unit lengths was similar, but not identical, to that observed previously for pol3-t, a temperature-sensitive mutation affecting DNA polymerase delta. Strains with both pol30-52 and pol3-t mutations grew extremely slowly and had minisatellite mutation rates considerably greater than those observed in either single mutant strain.

Conversion-type and restoration-type repair of DNA mismatches formed during meiotic recombination in Saccharomyces cerevisiae.

Meiotic recombination in yeast is associated with heteroduplex formation. Heteroduplexes formed between nonidentical DNA strands contain DNA mismatches, and most DNA mismatches in wild-type strains are efficiently corrected. Although some patterns of mismatch correction result in non-Mendelian segregation of the heterozygous marker (gene conversion), one predicted pattern of correction (restoration-type repair) results in normal Mendelian segregation. Using a yeast strain in which a marker leading to a well-repaired mismatch is flanked by markers that lead to poorly repaired mismatches, we present direct evidence for restoration-type repair in yeast. In addition, we find that the frequency of tetrads with conversion-type repair is higher for a marker at the 5' end of the HIS4 gene than for a marker in the middle of the gene. These results suggest that the ratio of conversion-type to restoration-type repair may be important in generating gradients of gene conversion (polarity gradients).