STRUCTURE AND BEHAVIOR OF YEAST TELOMERES

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Saccharomyces telomeres are essential for chromosome maintenance, at least in part because they protect chromosomes from degradation. Telomeres are also required to ensure the complete replication of the chromosome and possibly to help position chromosomes within the nucleus. Telomere structural proteins probably influence each of these functions. The goal of this grant is to identify structural proteins that interact with yeast telomeres and to determine their in vivo functions. The first aim is to develop a one-hybrid system for detecting telomere-protein interactions. This system will be used both to identify new telomere structural proteins and to assess whether known gene products that affect telomeres in vivo do so by interacting directly with telomeric DNA or telomeric chromatin. The second aim is to identify terminus-specific DNA binding proteins, that is proteins that bind only to telomeres, not to internal tracts of telomeric sequence. Genetic evidence suggests that such proteins influence telomere position effect, ThE, the ability of telomeres to repress transcription of nearby genes. Two independent approaches are proposed to identify terminus binding proteins. The biochemical approach is based on the fact that DNA termini in some organisms have short G-strand tails. A 15 kDa protein that binds to single-strand TG1-3 DNA in vitro will be purified, as the first step in isolating and characterizing the gene encoding it. The second approach is based on the observation that extra telomeres reduce ThE in trans, presumably by competing with chromosomal telomeres for terminus binding protein(s). Library plasmids that restore ThE to cells carrying extra telomeres will be isolated and characterized. Genetic evidence suggests that there is a factor that binds to the carboxyl terminus of telomere- bound Raplp whose depletion is associated with telomere lengthening and a cold sensitive (cs) growth phenotype. The third aim is to identify the gene encoding this factor by selecting library plasmids that reverse the cs and telomere length phenotypes and to determine how this protein limits telomere length. The fourth aim is to continue analysis of a collection of tst (telomere stability) mutants that affect expression of genes near telomeres, focusing on those mutants that affect telomere length and ThE. The fifth aim is to determine if telomeres are localized in a specific sub-compartment of the nucleus and, if so, if this localization is important for TPE. In yeast, telomere length and hence telomere function is controlled by the balanced activities of many genes, some that promote shortening and others lengthening of telomeric DNA. In humans, telomere length decreases with age. There is increasing speculation that the kinds of genetic instability associated with human disease, such as cancer and aging, could be triggered by the loss of telomeric DNA. Telomeric regions are structurally and functionally similar from yeast to humans. An understanding of the requirements for maintaining the structure and function of yeast telomeres is likely to be relevant to an understanding of the sources of genetic instability in humans.