Telomeres, the ends of eukaryotic chromosomes, are essential for the complete replication and stable maintenance of Saccharomyces chromosomes. Telomeres protect chromosomes from degradation, help the cell distinguish intact from broken DNA, provide a substrate for telomerase, and affect the transcription of nearby genes. The overall goal of this project is to identify structural proteins that interact with telomeres and to determine how they contribute to telomere function. The first goal is to exploit a one hybrid system for telomere binding proteins that was developed in the last funding period. The one hybrid assay will be used to identify new telomere binding proteins and to determine the binding requirements of known proteins. The second goal is to continue analysis of Cdc13p, a protein shown in the last funding period to bind single strand (ss) TG1-3 telomeric DNA in vitro and telomeres in vivo. The working model is that the physical presence of Cdc13p limits the cell cycle regulated degradation of C-strand telomeric DNA and recruits telomerase to the telomere. To test this hypothesis, biochemical and genetic approaches will be used to determine if the interaction of Cdc13p with Pollp and Estlp, as detected by two hybrid experiments, regulates either C-strand degradation or telomerase replication. The sites of Cdc13p phosphorylation will be determined and then mutated to assess the importance of phosphorylation to Cdc13p function. Chromatin immuno-precipitation (ChIP) will be used to determine if Cdc13p associates with the short ss tails thought to be present throughout the cell cycle and/or the long transient tails present at the end of S phase.
The third aim i s to determine how the large subunit of chromatin assembly factor I (CAF-1) reduces the stability of transcriptional repression near telomeres (telomere position effect, TPE). ChIP and genetic approaches will be used to determine if histone acetylation is important for CAF-1 mediated inheritance of TPE and if Sir protein association with telomeric or sub- telomeric DNA is altered in cells lacking CAF-1.
The fourth aim i s to determine if an individual telomere whose transcriptional state is known is localized to a specific sub-compartment of the nucleus. It will be determined if localization is affected by the transcriptional state of the telomere or by mutations that reduce TPE. There is increasing speculation that telomeres affect both aging and tumorigenesis in humans. Telomeres are well conserved from yeast to humans. An analysis of yeast proteins that affect telomere structure or function is likely to be relevant to an understanding of genetic instability in humans.
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