REGULATION OF TELOMERE LENGTH

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Telomeres are the nucleoprotein required for the stability and complete replication of chromosome ends. Telomere DNA consists of tandem arrays of TG-rich sequences: 10-20 kb of TTAGGG in humans and 250-400 bp of TG1-3 in budding yeast, where the TG-rich strand forms the 3' end of the chromosome. The length of these repeats is nearly constant in human germ cells and yeast, and is probably maintained by regulating the processes of lengthening, via telomerase, and shortening, due to incomplete replication or nuclease action. How these two processes are regulated to give a constant telomere length is unknown. In human somatic cells, the length of the TTAGGG tract decreases as cells divide, leading to cell senescence when telomeres become too short. How telomere length information is transmitted to the cell cycle machinery is unknown. Our long term goal is to use yeast as a model system to understand these processes. Yeast measure telomere length by counting the number of molecules of the major telomere binding protein Rap1p; however, how Rap1p molecules are counted is unknown. We developed a working model for telomere length regulation based on our construction of yeast synthetic telomeres and on our work with a telomere length regulator, TEL2. We propose that yeast telomeres form a folded structure with Rap1p and 2 regulatory proteins, Rif1p and Rif2p, to count Rap1p molecules and block elongation. Short telomeres have too few Rap1p molecules to form this structure, so they are elongated. Structure formation may be linked to a telomere-specific Rap1p modification that we have detected. TEL2 protein regulates yeast telomere length in vivo and binds to telomeric TG1-3 in vitro. TEL2 is in the same genetic pathway as TEL1, a homolog of the mammalian kinase DNA-PKcs and the yeast DNA damage checkpoint regulator of the cell cycle, MEC1. The morphology of cells lacking TEL2 suggests they arrest in a specific phase of the cell cycle. Thus, TEL2 may function to somehow link telomeres to cell cycle control. Our specific aims are to test our hypotheses that 1) telomere length is regulated by forming the folded structure we propose; 2) the telomere-specific modification of Rap1p is involved in telomere length control; and 3) Tel2p binds to telomeres in vivo and links telomeres to the cell cycle machinery. Our results will provide a model for how cells measure telomere length.