Thursday, November 21, 2024
11/21/2024
Project Summary/Abstract This R50 proposal is a companion to our recently renewed R35 (CA210036-08), which is focused on the role of telomeres in cancer. Telomeres are required for the survival and proliferation of human cells and play a critical role in cancer. Excessively long telomeres at birth predispose to a wide variety of cancers, presumably because long telomeres delay the Hayflick limit to a stage in tumorigenesis when incipient cancers have already disabled the cell cycle arrest response to short telomeres. Conversely, in the short telomere syndromes (e.g., dyskeratosis congenita and Coats plus syndrome) loss of telomeric DNA curbs the vitality of stem-cell compartments and instigates multi-organ failure. This proposal focuses on CST, the trimeric ssDNA-binding complex composed of Ctc1, Stn1, and Ten1 and its associated Pola/primase. CST– Pola/primase has a dual role at telomeres: it mediates maintenance of the telomeric C-strand and it regulates telomerase, preventing the excessively long telomeres that lead to cancer predisposition. As described in the preliminary data, we recently discovered a new end-replication problem that is not solved by telomerase. This problem arises from the inability of the replisome to sustain lagging-strand synthesis when it reaches the end of a linear DNA, a phenomenon we demonstrated using in vitro DNA replication in collaboration with Dr. Joseph Yeeles (LMB, Cambridge). I showed that the C-strand of telomeres shortens by ~60 nt during lagging-strand DNA synthesis in vivo and that the CST–Pola-primase complex is required to counteract this shortening. My additional preliminary data showed how CST–Pola- primase is recruited to telomeres. In collaboration with graduate student Sarah Cai, I demonstrated that CST binds to the shelterin subunit POT1, not TPP1, as had been anticipated. We derived the Cryo-EM structure of POT1 bound to CST and found that POT1 must be phosphorylated to recruit the complex to telomeres. These pathbreaking findings form the basis of my current proposal to determine how recruitment of CST– Pola/primase is regulated. My aims are to identify the kinase that phosphorylates POT1 to allow CST binding, study its regulation at telomeres, determine how dephosphorylation of POT1 releases CST–Pola/primase into the active fill-in complex, and how CST controls telomerase. I am a long-term member of the Unit Director’s group and a pivotal contributor to our R35 program. I have developed new technologies that are essential for our research. I am highly skilled, rigorous, creative, collaborative, and completely committed to the Unit Director’s research on the role of telomeres in cancer. My career goal is to continue to excel and support the success of the R35 through innovative research that delivers breakthrough discoveries.
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