Thomas D. Petes

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.

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.

Chromosomal Translocations in Yeast Induced by Low Levels of DNA Polymerase: A Model for Chromosome Fragile Sites

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.

Unequal meiotic recombination within tandem arrays of yeast ribosomal DNA genes

Recombinant DNA procedures and the yeast transformation technique were used to insert the yeast gene LEU 2 (coding for beta-isopropylmalate dehydrogenase) into the tandem array of ribosomal DNA genes of the yeast Saccharomyces cerevisiae. These insertions were shown by genetic and physical techniques to be unstable in meiosis. The meiotic instability was found to be the result of a high level of unequal recombination between ribosomal DNA gene clusters located on sister chromatids.

Interactions of TLC1 (Which Encodes the RNA Subunit of Telomerase), TEL1, and MEC1 in Regulating Telomere Length in the Yeast Saccharomyces cerevisiae

ABSTRACT In the yeast Saccharomyces cerevisiae, chromosomes terminate with a repetitive sequence [poly(TG1–3)] 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 inMEC1 (a gene related in sequence to TEL1 andATM) reduces telomere length and that tel1 mec1double 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 andtlc1 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.

Instability of simple sequence DNA in Saccharomyces cerevisiae.

All eukaryotic genomes thus far examined contain simple sequence repeats. A particularly common simple sequence in many organisms (including humans) consists of tracts of alternating GT residues on one strand. Allelic poly(GT) tracts are often of different lengths in different individuals, indicating that they are likely to be unstable. We examined the instability of poly(GT) and poly(G) tracts in the yeast Saccharomyces cerevisiae. We found that these tracts were dramatically unstable, altering length at a minimal rate of 10(-4) events per division. Most of the changes involved one or two repeat unit additions or deletions, although one alteration involved an interaction with the yeast telomeres.

Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome.

rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair. We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain. RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.

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.

Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)

ABSTRACT We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase δ) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both therad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the “proofreading” exonuclease domain of DNA polymerase δ) mutations were synthetically lethal.