Patricia W. Greenwell

TEL1, a gene involved in controlling telomere length in S. cerevisiae, is homologous to the human ataxia telangiectasia gene

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.

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.

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.

Caenorhabditis elegans DNA mismatch repair gene msh-2 is required for microsatellite stability and maintenance of genome integrity

Mismatch repair genes are important in maintaining the fidelity of DNA replication. To determine the function of the Caenorhabditis elegans homologue of the MSH2 mismatch repair gene (msh-2), we isolated a strain of C. elegans with an insertion of the transposable element Tc1 within msh-2. Early-passage msh-2 mutants were similar to wild-type worms with regard to lifespan and meiotic chromosome segregation but had slightly reduced fertility. The mutant worms had reduced DNA damage-induced germ-line apoptosis after genotoxic stress. The msh-2 mutants also had elevated levels of microsatellite instability and increased rates of reversion of the dominant unc-58(e665) mutation. In addition, serially passaged cultures of msh-2 worms died out much more quickly than those of wild-type worms. These results demonstrate that msh-2 function in C. elegans is important in regulating both short- and long-term genomic stability.

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.

High-Resolution Genome-Wide Analysis of Irradiated (UV and γ-Rays) Diploid Yeast Cells Reveals a High Frequency of Genomic Loss of Heterozygosity (LOH) Events

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.

Chromosome rearrangements via template switching between diverged repeated sequences

Recent high-resolution genome analyses of cancer and other diseases have revealed the occurrence of microhomology-mediated chromosome rearrangements and copy number changes. Although some of these rearrangements appear to involve nonhomologous end-joining, many must have involved mechanisms requiring new DNA synthesis. Models such as microhomology-mediated break-induced replication (MM-BIR) have been invoked to explain these rearrangements. We examined BIR and template switching between highly diverged sequences in Saccharomyces cerevisiae, induced during repair of a site-specific double-strand break (DSB). Our data show that such template switches are robust mechanisms that give rise to complex rearrangements. Template switches between highly divergent sequences appear to be mechanistically distinct from the initial strand invasions that establish BIR. In particular, such jumps are less constrained by sequence divergence and exhibit a different pattern of microhomology junctions. BIR traversing repeated DNA sequences frequently results in complex translocations analogous to those seen in mammalian cells. These results suggest that template switching among repeated genes is a potent driver of genome instability and evolution.

Genome-wide high-resolution mapping of chromosome fragile sites in Saccharomyces cerevisiae

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). In mammalian cells, perturbations in DNA replication result in chromosome breaks in regions termed “fragile sites.” Using DNA microarrays, we mapped recombination events and chromosome rearrangements induced by reduced levels of the replicative DNA polymerase-α in the yeast Saccharomyces cerevisiae. We found that the recombination events were nonrandomly associated with a number of structural/sequence motifs that correlate with paused DNA replication forks, including replication-termination sites (TER sites) and binding sites for the helicase Rrm3p. The pattern of gene-conversion events associated with cross-overs suggests that most of the DNA lesions that initiate recombination between homologs are double-stranded DNA breaks induced during S or G2 of the cell cycle, in contrast to spontaneous recombination events that are initiated by double-stranded DNA breaks formed prior to replication. Low levels of DNA polymerase-α also induced very high rates of aneuploidy, as well as chromosome deletions and duplications. Most of the deletions and duplications had Ty retrotransposons at their breakpoints.

Genome rearrangements caused by interstitial telomeric sequences in yeast

Significance Telomeres are composed of simple repetitive DNA sequences that normally are located at the ends of the chromosomes. Occasionally, however, they also are found inside chromosomes. Some of these internal or interstitial telomeric sequences colocalize with chromosomal fragile sites, preferred sites of breakage in some cancers and hereditary human diseases. The mechanisms responsible for genome instability at interstitial telomeric sequences are unclear. We developed a system to study genetic instabilities caused by these sequences in a model organism (baker’s yeast) that allowed us to characterize various chromosomal rearrangements and to measure the likelihood of their formation. We found that interstitial telomeric sequences promote the formation of deletions, duplications, inversions, and translocations, and we proposed molecular mechanisms responsible for these events. Interstitial telomeric sequences (ITSs) are present in many eukaryotic genomes and are linked to genome instabilities and disease in humans. The mechanisms responsible for ITS-mediated genome instability are not understood in molecular detail. Here, we use a model Saccharomyces cerevisiae system to characterize genome instability mediated by yeast telomeric (Ytel) repeats embedded within an intron of a reporter gene inside a yeast chromosome. We observed a very high rate of small insertions and deletions within the repeats. We also found frequent gross chromosome rearrangements, including deletions, duplications, inversions, translocations, and formation of acentric minichromosomes. The inversions are a unique class of chromosome rearrangement involving an interaction between the ITS and the true telomere of the chromosome. Because we previously found that Ytel repeats cause strong replication fork stalling, we suggest that formation of double-stranded DNA breaks within the Ytel sequences might be responsible for these gross chromosome rearrangements.

Gene Copy-Number Variation in Haploid and Diploid Strains of the Yeast Saccharomyces cerevisiae

The increasing ability to sequence and compare multiple individual genomes within a species has highlighted the fact that copy-number variation (CNV) is a substantial and underappreciated source of genetic diversity. Chromosome-scale mutations occur at rates orders of magnitude higher than base substitutions, yet our understanding of the mechanisms leading to CNVs has been lagging. We examined CNV in a region of chromosome 5 (chr5) in haploid and diploid strains of Saccharomyces cerevisiae. We optimized a CNV detection assay based on a reporter cassette containing the SFA1 and CUP1 genes that confer gene dosage-dependent tolerance to formaldehyde and copper, respectively. This optimized reporter allowed the selection of low-order gene amplification events, going from one copy to two copies in haploids and from two to three copies in diploids. In haploid strains, most events involved tandem segmental duplications mediated by nonallelic homologous recombination between flanking direct repeats, primarily Ty1 elements. In diploids, most events involved the formation of a recurrent nonreciprocal translocation between a chr5 Ty1 element and another Ty1 repeat on chr13. In addition to amplification events, a subset of clones displaying elevated resistance to formaldehyde had point mutations within the SFA1 coding sequence. These mutations were all dominant and are proposed to result in hyperactive forms of the formaldehyde dehydrogenase enzyme.