Date of Award
de Lange Laboratory
telomeres, telomeric overhang, Apollo, Exo1, CST complex
The telomeric single-stranded 3â€™ overhang, a conserved feature at the ends of linear chromosomes, is thought to contribute to the important functions of end protection and telomere length homeostasis. Here, I investigated the mechanism by which the overhang is generated and maintained at mammalian telomeres. First, I evaluated the terminal chromatin structure of mouse telomeres using conventional micrococcal nuclease (MNase) assays and a novel method to examine the terminal nucleosomes adjacent to the telomeric overhang. In wild type mouse embryonic fibroblasts (MEFs), the telomeric overhang was protected from MNase digestion of chromatin in nuclei. In addition, the induction of various DNA damage responses at telomeres, through the deletion of shelterin components, had no apparent effect on the chromatin organization of telomeres and did not cause overt eviction of nucleosomes from the telomere terminus. Next, I identified a role for the shelterin-associated Apollo nuclease in generating the telomeric overhang at ends synthesized by leading-strand DNA replication. Deletion of Apollo in mouse cells resulted in an overhang defect specifically at leading-end telomeres. Consistent with a requirement for the overhang in end protection, cells lacking Apollo activated an ATM-dependent DNA damage response at a subset of telomeres in S phase and displayed fusions between a fraction of leading-end telomeres on metaphase spreads. I next elucidated the mechanism by which the single-stranded telomere binding protein, POT1b, suppresses the accumulation of excessive telomeric overhangs in mouse cells. I found that POT1b inhibits 5â€™ resection by Apollo at telomeres synthesized by both leading- and lagging-strand DNA replication while promoting the telomeric function of the Ctc1/Stn1/Ten1 (CST) complex. Though the role of CST is not well understood, Ctc1 and Stn1 were originally identified as accessory factors of DNA polymerase Î±/primase and may contribute to fill-in synthesis of the telomeric C-rich strand. Finally, to identify additional factors that contribute to overhang dynamics, I tested the hypothesis that the factors involved in 5â€™ end resection at a DNA double strand break also act at telomeres in wild type cells. I uncovered a role for exonuclease 1 in a transient telomere end-processing step that occurs at both newly-synthesized telomeres. The Exo1-mediated telomere end-processing step does not appear to require Nbs1 or BLM, and its physiologic role remains unknown. This thesis supports a model in which leading- and lagging-end telomeres have different requirements for end-processing by the nucleases Apollo and Exo1. The binding of POT1b on appropriately generated single-stranded overhangs at both newlysynthesized telomeres limits overhang size and prevents accelerated telomere shortening by inhibiting Apollo and facilitating the activities of the CST complex.
Wu, Peng, "Mechanisms That Ensure the Proper Terminal Structure of Mammalian Telomeres" (2012). Student Theses and Dissertations. 141.