The goal of this proposal is to study the Ku heterodimer's (Ku70/80) role in telomere maintenance and cellular senescence in mammals. Support for Ku's involvement in telomere maintenance is provided by the recent demonstration that the Ku homologs of yeast are required for telomere maintenance. Moreover, it has been shown that yeast Ku is in close proximity to telomeric DNA in vivo, suggesting a direct role for Ku in telomere length regulation. Strikingly, despite the relatively low sequence conservation between yeast and human Ku subunits at the amino acid level, the exogenous expression of human Ku subunits in yeast rescues a Ku null mutation. In this proposal, the applicant presents recently obtained novel preliminary evidence for an association of Ku with telomeric DNA in human cells. Telomere maintenance is critical for senescence, since activation of telomerase, the enzyme responsible for the synthesis of telomeric DNA, has been shown to directly control replicative senescence in primary human cell. The applicant proposes a detailed investigation into the interactions between known elements of the telomere and Ku to determine the role played by these proteins in telomere maintenance and replicative senescence. The applicant's specific aims are to investigate: 1) Ku/telomeric DNA interactions at the mammalian telomere using in vivo and in vitro experimental approaches; 2) whether known mammalian telomere binding proteins mediate an interaction between Ku and the telomere; and 3) telomere maintenance in embryonic stem cells and cellular senescence in mouse embryonic fibroblasts with various mutations in Ku70, Ku 80, DNA-PKcs and the RNA moiety of telomerase. This proposal brings together two groups with complementary expertise: Dr. Elizabeth Blackburn's group at the University of California, San Francisco with expertise in the telomere and telomerase, and Dr. David Chen's group at Los Alamos National Lab with expertise in the Ku heterodimer and DNA-PKcs. The goal of this work is to form an understanding of the interaction of these key components, the telomere and the Ku heterodimer, in mammalian cells. These studies are critical for a further understanding of telomere maintenance, which will provide a better understanding of cellular senescence.