PROJECT SUMMARY Without a dedicated mechanism to replicate their ends, human chromosomes shorten with each cell division, leading to senescence and death. A specialized RNA-protein enzyme complex, telomerase, counteracts this end-replication problem, lengthening telomeres to complete genome duplication. Telomerase becomes dormant during development of human tissues, leading to chromosome shortening with age. However, it reemerges in most human cancers, sustaining cell proliferation potential. Basic information is needed about how telomerase operates to maintain telomeres in order to develop therapies that control these process. Studies indicate that telomere length homeostasis is achieved by telomerase being preferentially recruited to the shortest telomeres, yet the system that controls this is not understood. In yeast, two pathways have been identified for telomerase recruitment, based on its Ku and Est1 subunits. This project seeks to identify how telomerase is selectively recruited to short telomeres to lengthen them. The research evaluates the hypotheses that (1) the Ku and Est1 telomerase-recruiting pathways operate in a concerted, stepwise manner and (2) that the telomeric Rif proteins regulate telomere length by antagonizing each recruitment step by specific independent mechanisms that weaken as telomere length shortens. Aim 1 will first use a new single-telomere system to define the length of a ?short? telomere and each telomeric component functions to stimulate or suppress telomerase recruitment and action. The second goal of Aim 1 is to pinpoint where telomeric proteins and telomerase associate with DNA along the distal portions of chromosomes, using a deep-sequencing- based assay that has near single-nucleotide resolution. This will advance our understanding of telomere structure and telomerase regulation by revealing the arrangement of telomeric proteins along telomeric repeats and identifying where telomerase is recruited relative to chromosome ends. Aim 2 will determine how close to an individual chromosome end telomerase needs to be recruited to extend it and also test if the Ku pathway relies on Est1 to promote telomerase recruitment and activity. Finally, Aim 3 will investigate how telomerase, once recruited, accesses and extends a chromosome end by examining an essential, conserved telomerase RNA moiety recently found to be required after enzyme recruitment. The experiments will identify the proteins that bind telomerase RNA by an RNA-protein two-hybrid interaction assay as well as more conventional approaches. Overall, this project will provide critical mechanistic insights into how cells maintain telomere length in the major yeast model organism and establish the framework to understand telomere length regulation in humans. Given the critical function of telomere maintenance in sustaining cell growth potential in cancer and aging, learning how telomerase maintains telomeres will have a major impact on improving human health by revealing the best molecular targets for therapeutics.