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Characterization of the role of SUMO in telomere length homeostasis and overhang processing at yeast telomeres

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posted on 2023-06-09, 06:45 authored by Mansi Garg Aggarwal
Telomeres protect the ends of the linear chromosomes by assembling a nucleoprotein complex called shelterin. This complex regulates the action of telomerase, a reverse transcriptase enzyme that adds telomeric DNA to the G-rich 3’ end of the chromosomes. CST (CTC1/Cdc13-Stn1-Ten1) complex, also associated with telomeres, counteracts telomerase mediated telomere overhang elongation by recruiting lagging strand DNA polymerases for C-Strand synthesis. SUMOylation, a post-translational modification, is known to contribute towards negative regulation of telomerase in both budding yeast and fission yeast. It has previously been shown in budding yeast that SUMOylation of Cdc13 enhances its interaction with Stn1 to restrain telomerase function. In humans, CST interacts with overhang-binding shelterin proteins TPP1/POT1, but there is no evidence whether this interaction is regulated. The work in this thesis provides a mechanistic insight into the role of SUMOylation in telomere length regulation in fission yeast. It was established in this study that fission yeast telomeric protein Tpz1, an ortholog of human TPP1, is SUMOylated at lysine 242 by SUMO E3 ligase Pli1. The mutation of this lysine residue leads to telomere elongation in a telomerase-dependent manner. Chromatin immunoprecipitation (ChIP) analysis indicated that the association of telomerase with telomeres is increased in a tpz1-K242R mutant, whereas that of Stn1 and Ten1 is greatly reduced. In addition, a SUMO-Tpz1 fusion protein showed increased affinity for Stn1, in the yeast two-hybrid assays. The assay was also used to define minimal region in Tpz1 required for its interaction with Stn1. This data indicated that SUMOylation of Tpz1 facilitates the recruitment and function of Stn1-Ten1 at telomeres, which in turn down-regulates telomerase. Collectively, this study highlight the evolutionary conservation of the regulation of (C)ST function by SUMOylation and provides further insight into the role of Tpz1 in telomerase regulation. In order to obtain further insight into the dynamics of telomerase action and overhang fill-in by C-strand synthesis I also developed and validated a PCR-based assay for precise measurement of telomere overhang i budding and fission yeast.


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University of Sussex

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