Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

Telomeres are the nucleoprotein complexesrequired for the stability and completereplication of chromosome ends. Brokenchromosome ends undergo permanent fusions toother DNA molecules in the cell or are degraded,resulting in genomic rearrangements that can leadto cancer. In contrast to broken ends, telomeresare stable and preserve the cell’s genomicintegrity.Telomere DNA consists of tandem arraysof TG-rich sequences: 10-20 kb of TTAGGG inhuman germ cells and 250-400 bp of TG 1-3inbudding yeast, where the TG-rich strand formsthe 3' end of the chromosome. The length ofthese repeats is nearly constant in human germcells and yeast and is probably maintained byregulating the processes of lengthening viatelomerase (a special enzyme that can synthesizetelomere repeats) and shortening caused byincomplete replication or nucleolytic processingof telomeric DNA.How these two processes are regulated tomaintain telomere lengths within a defined rangeis unknown. In human somatic cells, the lengthof the TTAGGG tract decreases as cells divide,leading to cell senescence when telomeresbecome too short. How telomere lengthinformation is transmitted to the cell-cyclemachinery is unknown. Our long-term goal is touse yeast as a model system to understand theseprocesses.Yeast measures telomere length bycounting the number of molecules of the majortelomere binding protein Rap1p; however, howRap1p molecules are counted is unknown. We(Ray and Runge, 1999a,b) developed a workingmodel for telomere length regulation based onour construction of yeast synthetic telomeres andon our work with the telomere length regulatorTEL2. We propose that yeast telomeres form afolded structure with Rap1p, Rif1p and Rif2p tocount Rap1p molecules andblock elongation. Shorttelomeres have too few Rap1pmolecules to form thisstructure, so they are elongated.We (Kota and Runge,1999) have found that theprotein encoded by TEL2(Tel2p) regulates yeast telomerelength in vivo and binds totelomeric TG 1-3in vitro. TEL2 isin the same genetic pathway asTEL1, a homolog of themammalian kinase DNA-PKcsand the yeast DNA damagecheckpoint regulator of the cellcycle, MEC1. The morphologyof cells lacking TEL2 suggeststhat they arrest in a specificThe Department of Molecular BiologyTelomere Length Regulation,Transcriptional Silencing and Cell Agingphase of the cell cycle. Thus, TEL2 may functionto link telomeres to cell-cycle control.We (Roy and Runge, 2000) have recentlydiscovered conditions that cause Sir proteins to beredistributed in the cell in a manner that extendscell life span. Because this redistributioncorrelated with Sir3p phosphorylation, weidentified the Sir3p kinase as Slt2p (a yeast MAPkinase), eliminated Sir3p phosphorylation byeliminating the predicted phosphorylation sites,and showed that lack of Sir3p phosphorylationcauses an increase in cell life span. Because theMAP kinase that phosphorylates Sir3p is activatedby the cell’s decision to grow, the Sir3p systemforms a model for the known phenomenon oforganisms exhibiting slow reproduction andlonger life span understarvation conditions,and more reproudctionand shorter life spanwhen nutrients areplentiful.THE RUNGELABORATORYRESEARCH ASSOCIATESRonald Hector, Ph.D.Alo Ray, Ph.D.Ken richardson, M.D.TECHNICAL ASSOCIATESThihan NyunAnna YakubenkoKurt W. Runge, Ph.D.Kota, R.S., and K.W. Runge (1999) Tel2p, a regulator of yeast telomerre length in vivo, binds to singlestrandedtelomeric DNA in vitro. Chromosoma 108:278-290.Ray, A., and K.W. Runge (1999a) Varying the number of telomere bound proteins does not alter telomerelength in tel1? cells. Proceedings of the National Academy of Sciences USA 96:15044-15049.Ray, A., and K.W. Runge (1999b) The yeast telomere length counting machinery is critically sensitiveto sequences at the telomere/non-telomere junction. Molecular and Cellular Biology 19:31-45.Roy, N., and K.W. Runge (2000) Two paralogs involved in transcriptional silencing that antagonisticallycontrol yeast life span. Curr. Biol. 10:111-114.Ray, A., and K.W. Runge (2001) Yeast telomerase appears to frequently copy the entire template invivo. Nucleic Acids Res. 29:2382-2394.Ray*, A., Hector*, R.E., Roy, N., Song, J.H., Berkner, K.L., and K.W. Runge (2003) Sir3p phosphorylationby the Slt2p pathway effects redistribution of silencing function and shortened lifespan. Nat. Genet.33:522-526 (*co-first authors).109