The goal of our research is to define the molecular mechanism by which telomere attrition limits the proliferation of human cells. Replicative senescence (and apoptosis) can be caused by telomere attrition, implicating telomere dynamics in cellular aging. In order to understand this process, we have studied the protein components of human telomeres and found that the telomeric protein TRF2 is a main determinant of the protective state of telomeres. Inhibition of TRF2 results in a senescence phenotype that is indistinguishable from replicative senescence. Since TRF2 inhibition mimics replicative senescence, we propose that shortened telomeres in aged cells are dysfunctional because they contain insufficient TRF2. In agreement, overexpression of TRF2 allows cells to continue to divide with critically shortened telomeres, making TRF2 a candidate longevity assurance gene (LAG). TRF2 protects the telomeric 3' overhang and promotes the formation of the t-loop, a higher order structure proposed to protect telomeres. Therefore, loss of either the overhang and/or the t-loop may be what causes critically shortened telomeres to induce cell cycle arrest. We have also obtained evidence that the telomere damage signaling pathway is similar to the DNA damage response and is governed, in part, by the ATM kinase. Based on the binding of DNA damage response factors to uncapped telomeres, we have developed an assay that allows dissection of the telomere damage signaling pathway. In this proposal we will use TRF2 as a tool to dissect the main molecular determinants of the telomere damage signaling pathway. In AIM 1 we address which molecular change at the telomere activates the DNA damage response. We will test whether the loss of the telomeric overhang is the crucial signal, whether the opening of t-loops is the main event, or whether complete telomere loss occurs. In AIM 2, we will address the sensor(s) that detect the altered state of shortened telomeres. Specifically, we will ask whether the Mre 11 complex, RPA, and/or 9-1-1 are involved. Finally, in AIM 3, we will address how the telomere damage signal is transduced. We will determine the role of ATM, ATR, and DNA-PKcs in the detection of dysfunctional telomeres. These experiments should reveal how telomere attrition limits the replicative life-span of human cells. Given the mounting evidence supporting a role for telomere shortening in human aging, our findings will be relevant to human disease states. [unreadable] [unreadable]