The broad goal of this project is to gain insight in the function of mammalian telomeres. Telomeres are specialized elements at chromosome ends that are composed of an array of telomeric repeats and telomere-associated proteins. The telomeric nucleoprotein complex has at least two functions. First, it renders the natural end of chromosomes resistant to DNA repair enzymes and hides the telomere termini from cell cycle checkpoints. Second, the telomeric complex allows chromosome ends to interact with telomerase, an RNA-dependent DNA polymerase that can elongate the telomeric repeat array. Telomerase activity can make up for the gradual loss of terminal sequences that accompanies replication of linear DNAs. The principal objective of this study is to identify telomeric proteins at mammalian chromosome ends and to characterize the contribution of these proteins to telomere function. Telomeric proteins have been studied in detail in yeast and in ciliates but not in multicellular organisms. We have isolated and cloned a mammalian telomeric protein, the telomeric repeat binding factor, TRF. In this proposal, TRF is used as a tool to isolate additional telomere-associated proteins and to address the mechanism of telomere function in human and mouse cells. TRF may be required in the telomeric complex to hide chromosome ends from cell cycle checkpoints and repair functions. To test this idea, we propose to inactivate TRF and to evaluate cell cycle progression and chromosome instability in the absence of (normal) TRF activity. The possibility that TRF contributes to the regulation of telomere replication will be approached by analyzing telomere dynamics in cells with impaired TRF function and by in vitro assays for interactions of TRF with telomerase. These studies are expected to reveal how TRF participates in the protective role of telomeres and what the contribution is of TRF to the regulation of telomere maintenance. Telomere dynamics have been implicated in human cancer and aging. Together with the isolation of additional telomere associated proteins, the functional dissection of the mammalian telomeric complex proposed here should lead to a better understanding of the role of telomeres and telomerase in normal and malignant human cells.
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