Zhao, Y; (2012) Pot1 phosphorylation regulates telomere function. Doctoral thesis (PhD), UCL (University College London).

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Abstract

The telomere is a conserved nucleoprotein structure at the ends of eukaryotic chromosomes. It is essential for maintenance of genomic stability: on the one hand, it suppresses DNA damage response and protects the natural chromosome ends from repair activities; on the other hand, it recruits telomerase, the specialized reverse transcriptase, to counteract the end-replication problem. The telomeric G-strand ssDNA-binding protein Pot1 plays a crucial role in both of these functions. In fission yeast S. pombe, inhibition of Pot1 induces rampant 5’ resection and loss of telomere signal in a single cell cycle. It was recently shown that spPot1 interacts with, and is phosphorylated by, the master cell cycle regulator DDK. Alleles of a V5-tagged version of pot1+ were constructed with mutations at the putative phosphorylation sites, which reside in the N-terminal OB-fold DNA binding domain of Pot1 {Kuznetsov, 2008 #6380}. The goal of this study was to determine the molecular mechanism by which phosphorylation of Pot1 regulates telomere function. We found that the phospho-deficient mutants of Pot1 induce telomere elongation, checkpoint activation, and deregulation of ssDNA generation, suggesting reduced association with the ssDNA. Our data point to a model in which cell cycle-regulated Pot1 phosphorylation coordinates telomere replication and telomerase activity in different cell cycle phases. Furthermore, we showed that the C-terminal V5-tagging of Pot1 also affects its functions, suggesting an additional layer of complexity governing Pot1 function.

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