Thomas D. Petes - US grants

The long term goal of the proposed research is to understand the rules governing recombination between repeated eukaryotic genes; the organism that will be used in these studies is the yeast Saccharomyces cerevisiae. This organism is particularly useful for the proposed studies since the development of the yeast transformation procedure allows construction of haploid yeast strains with duplicated copies of selectable genes. Five specific areas of research will be pursued. First, the properties of recombination between repeated genes located on non-homologous chromosomes will be analyzed. The frequency of such events will be examined as a function of the size and location of the repeated sequences. Second, the frequency of recombination between repeated sequences located on the same chromosome will be studies. These experiments will be done in the same genetic background and with the same repeated sequences as the studies involving non-homologous chromosomes; thus, a direct measurement of the relative frequency of these types of events can be made. Third, a genetic system will be developed to look for duplication of genes normally found in one copy per genome. Such events are likely to be important in understanding the origin of repeated gene families. Fourth, mutant strains will be isolated which have either higher or lower rates of recombination between repeated genes. Fifth, the properties of recombination involving transposable elements will be characterized. Since recombination between repeated genes on non-homologous chromosomes can result in translocations and since the activation of cellular onc genes in mammals is often associated with translocations, these studies may lead to insights into health-related problems.

The purpose of the research is to determine what fraction of the DNA sequences of the yeast Saccharomyces cerevisiae are required for cell viability. The experimental approach is to construct in vitro gene disruptions of random segments of yeast DNA cloned on recombinant plasmids. These disrupted sequences will be transformed into a diploid yeast strain, creating a gene disruption on one of the two homologous chromosomes. The diploid strain will then be induced to undergo meiosis. Gene disruptions that result in recessive lethals will meiotically segregate two live spores to two dead spores. The fraction of random gene disruptions resulting in haploid lethality should be directly related to the fraction of yeast DNA sequences required for cell viability. In strains containing haploid-viable gene disruptions, attempts will be made to correlate other phenotypes to the disruption (such as effects on cell growth rate, temperature sensitivity, auxotrophic requirements, mating or sporulation defects and mutagen sensitivity). The transcriptional activity of the disrupted sequences will also be monitored. In addition to information about the fraction of yeast DNA sequence required for cell viability, the Southern analysis of the various insertions that are part of the study will give a good estimate of the fraction of yeast DNA sequences that are repeated (as well as the repetition frequency of each family of repeats). The yeast strains containing haploid-viable insertions will be used in subsequent studies of the effects of flanking DNA sequences on gene expression and recombination.

The yeast Saccharomyces cerevisiae, like other eukaryotes, contains linear chromosomes. The long-term goal of the proposed research is to establish the structure chromosomes. The long-term goal of the proposed research is to establish the structure and mode of replication of the ends (telomeres) of the yeast chromosome. Since all known DNA polymerases elongate DNA in a 5' to 3' direction and require a primer to initiate replication, it is clear that the telomeres cannot be replicated by a conventional replication mechanism. In previous studies, it has been observed that yeast chromosomes terminate in a simple sequence that can be abbreviated polyC1-3A and that yeast cells have a mechanism that allows the untemplated addition of polyC1-3A sequences to certain linear substrates. Since it is likely that this mechanism is related to telomere replication, most of the proposed experiments are concerned with elucidating the details of the terminal addition reaction. Both genetic and physical studies of the reaction will be done. Yeast mutants that add tracts of polyC1-3A that are longer (or shorter) than normal will be isolated and characterized. The physiological conditions that influence tract length in the cell and the substrate sequences necessary to mediate the terminal addition of polyC1-3A in vivo will be examined. In addition, experiments designed to look for de novo formation of telomeres on the ends of broken chromosomes are discussed. A system that mediates the in vitro addition of terminal polyC1-3A sequences will be developed. Since all eukaryotes contain linear chromosomes, it is likely that the proposed studies will have general relevance. In addition, since the terminal addition of polyC1-3A residues to linear DNA molecules is a previously unobserved modification of DNA, the mechanism involved in the modification deserves further analysis.

DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanisms of meiotic and mitotic recombination. A knowledge of the mechanisms by which cells create double-stranded DNA breaks (the DNA lesions that initiate recombination) and repair these breaks is essential for understanding the genomic instability associated with cancer. Although most of our proposed experiments will be done with the yeast Saccharomyces cerevisiae, we will examine DNA mismatch repair (MMR) in both yeast and C. elegans. The first Specific Aim is to examine the properties of DNA sequence and chromatin structure that affect the frequency of meiotic recombination. For most of these experiments, we will use DNA microarrays to monitor meiotic recombination activity at every locus in the genome. The second Specific Aim is to study the mechanism of reciprocal mitotic recombination. Using a system that allows selection of reciprocal mitotic crossovers, we will determine whether there are hotspots for mitotic exchanges. We will also investigate the genetic regulation of mitotic recombination using mutations that affect various pathways of recombination and DNA repair. Our third Specific Aim is to examine various aspects of DNA mismatch repair in yeast and in C. elegans. We will analyze the roles of the yeast MMR proteins Msh3p, Msh6p, MIh2p, and MIh3p, as well as the possible involvement of the DNA replication proteins DNA polymerase delta and PCNA, in the repair of mismatches generated during meiotic recombination. The proposed C. elegans studies will concentrate on determining phenotypes associated with msh-2 and msh-6 mutant worms, and on the isolation of new MMR worm mutants.

The long-term objective of the proposed research is to understand recombination events that involve little or no DNA sequence homology. Such events have been detected in a number of ways. For example, when mammalian cells are transformed with DNA, the transforming sequences usually integrate into the chromosome by a non-homologous recombination event. In contrast, transforming DNA in the yeast Saccharomyces cerevisiae has been reported to integrate into the genome only by homologous recombination. Recently, integration of transforming DNA in yeast by non-homologous recombination has been observed. In this proposal, the rules governing this type of recombination will be examined. In particular, the genetic control of this type of exchange will be investigated by selecting mutants with higher (or lower) levels of illegitimate recombination. It will also be determined whether the illegitimate integration of transforming DNA is related to the formation of chromosomal deletions in yeast. Since illegitimate integration events are a serious problem in the development of protocols for gene therapy, these studies should be relevant to health-related problems. A second area of proposed research is the analysis of recombination events catalyzed by exogenously-added restriction enzymes. The use of this technique for insertional mutagenesis, and for the creation of chromosomal aberrations will be examined.

DESCRIPTION: Microsatellites are regions of DNA in which a single base or a small number of bases is repeated multiple times. All eukaryotes examined thus far contain microsatellites. These sequences are inherently unstable, undergoing alterations in length at a much higher rate than that observed for "normal" DNA sequences. Genome-wide microsatellite instability is associated with certain types of human cancer. In yeast, mutations in DNA mismatch repair genes result in microsatellite instability and some human tumors with unstable microsatellites have mutations in human homologues of the yeast DNA mismatch repair genes. Some microsatellite unstable tumors, however, lack such mutations. In the current proposal, mutant screens designed to identify new genes affecting microsatellite instability in yeast are proposed. If new genes are identified, human homologues of these genes will be isolated. DNA isolated from tumors with unstable microsatellites that do not have mutations in known DNA mismatch repair genes will be examined to determine whether these tumors have mutations in human homologues of the newly-discovered yeast genes. Three other types of experiments concerning microsatellite instability are proposed. Since mistakes made during DNA replication are likely to contribute to microsatellite instability, a search for mutations in yeast DNA polymerase delta that result in unstable microsatellites will be performed. Second, the genetic regulation of microsatellite in mitochondrial DNA in yeast will be studied. Third, a transgenic mouse line will be constructed to allow the measurement of microsatellite instability in vivo.

DESCRIPTION (provided by applicant): In mammals, two related genes, ATM and AIR, encode very large protein kinases involved in the maintenance of genome stability. Patients with mutations in ATM develop the neurodegenerative disease ataxia telangiectasia (AT). AT patients are very cancer-prone, and cells derived from AT patients are very sensitive to DNA damaging agents. In addition, chromosomes of AT cells have short telomeres; hyper-recombination and chromosome breakage are also associated with mutations in ATM and ATR. The yeast Saccharomyces cerevisiae has two genes, TELl and MEC1 that are structurally and functionally related to ATM and ATR. Yeast strains with tell mec1 mutations have genetic instability that mimics that observed in mammalian cells that have mutations in ATM or ATR: high frequencies of chromosome rearrangements and chromosome loss, a strong mutator phenotype, loss of telomeric sequences, and cellular senescence. The general goal is to understand the genetic instability of tell mec1 strains. Some of the specific aims of this proposal are: 1) to characterize the mechanisms involved in generating chromosome aberrations in the tell mec1 strain; 2) to determine whether the production of chromosome aberrations is causally linked to cellular senescence; 3) to determine whether the Tell and/or Mec1 proteins affect telomere structure; 4) to define the biologically-relevant substrates of the Tell p and Mec1 p kinase activities; and 5) to use genetic screens to identify proteins that interact with Tell p and Mecip. For many of these studies, the phenotypes observed in tell med1 strains will be compared to those found in strains lacking telomerase, since strains lacking telomerase also undergo cellular senescence.

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area (08), "Genomics", and the specific Challenge Topic (08-ES-106), "The role of environmental exposure in copy number variation (CNV)". CNV events are deletions or duplications that are too small to be recognized by karyotypic analysis. Spontaneous CNVs have been recently recognized as a major source for sporadic disease in human populations, perhaps most notably in Autism Spectrum Disorders (ASD). The mechanisms underlying the formation of CNVs are not known, although it is likely that both environmental and intrinsic cellular factors affect the rate of CNVs. In this proposal, we describe the use of the yeast Saccharomyces cerevisiae as a model to find out how environmental contaminants and various mutations control the rate of CNVs. We will examine both large (>1kb deletions or duplications) and small (1- 1000 bp) CNV events. To investigate the effect of environmental factors and contaminants on large CNV events (Specific Aim 1), we will use a reporter in which duplications can be selected and deletions can be detected by screening. We will use a cassette that contains two yeast genes SFA1 and CUP1 that confer gene dosage-dependent tolerance to formaldehyde and copper, respectively. Using yeast strains in which this cassette is integrated into various positions, we will measure the rate of cassette amplifications in a wild-type strain growing under standard lab conditions. We will then determine the rate of amplifications in response to various DNA damaging agents, and common agrochemicals. We will also optimize a high-throughput version of the formaldehyde-copper resistance assay for use in preliminary screens of diverse chemical libraries to identify unknown environmental contaminants associated with CNV stimulation. To determine which mechanisms are involved in the amplifications (homologous recombination between dispersed repeats, microhomology- mediated recombination, non-homologous end joining or aneuploidy), we will characterize the strains with CNV events using DNA microarrays and a gel system that separates intact chromosomal DNAs (CHEF gels). In Specific Aim 2, we will conduct an investigation of cellular mechanisms linked to CNV formation, focusing on spontaneous events and those stimulated by low-dose ionizing radiation. We will determine the rates of CNV and the types of alterations in cells exposed to radiation at different stages of the cell cycle. In addition, we will examine spontaneous CNVs in meiotic cells. A set of mutant strains with compromised DNA repair and replication will also be examined to characterize the genetic control of CNV formation. In Specific Aim 3, we will investigate a different class of CNV events, those that duplicate or delete less than 1000 bp. For this analysis, we will use high-throughput DNA sequencing of sub-cultured untreated yeast strains and of strains treated with low levels of radiation. The analysis should provide much needed insight into the prevalence of this class of CNV, as well as into the mechanism of its formation. In this proposal, we examine the effects of environmental stresses and alterations of the genetic background on the frequency of de novo copy number variation (deletions and duplications of DNA sequences) in yeast. During the past few years, it has become increasingly clear that copy number variation (CNV) is associated with many human diseases. Thus, factors that elevate the rate of CNV are likely to elevate the frequency of those diseases.