The ends of linear chromosomes, called telomeres, are essential for genomic stability and normal cellular growth. In most organisms, the primary mechanism for complete replication of telomeres relies on the enzyme telomerase. Loss of telomerase function results in a progressive decline in telomere length that heralds replicative senescence, which has been postulated to contribute to organismal aging. This enzyme is also reactivated in the majority of human tumors, indicating that telomerase is a likely target for anti-cancer therapeutics. A full appreciation of the contribution of telomere replication to these two important aspects of human biology will require a molecular understanding of how telomerase is regulated in vivo. We are using the budding yeast, S. cerevisiae, as a system for dissection of this problem, based on the assumption that what we learn in yeast will translate to human cells. Past work in our laboratory has identified three proteins, Est1, Est2 and Est3, that are subunits of yeast telomerase. The Est2 protein is the catalytic reverse transcriptase component, whereas Est1 and Est3, which are dispensable for enzyme catalysis but essential in vivo for telomere replication, are positive regulatory subunits of the telomerase holoenzyme. Our genetic and biochemical analysis has shown that the Est1 and Est3 subunits contribute multiple, and functionally distinct, regulatory roles to telomere replication. We have also characterized potential regulatory domains of the TLC1 RNA subunit of the yeast telomerase enzyme. Additional recent studies have led to the identification of additional genes required for regulation of telomere replication, including a potential negative regulator of telomerase. In this application, we propose a set of experiments intended to form an integrated picture of how these positive and negative regulators of telomerase control telomere replication.
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