The functional evolution of telomere proteins in Tetrahymena thermophila

The functional evolution of telomere proteins in Tetrahymena thermophila

Bibliographic Details
Main Author: Cranert, Stacey
Language:English
Published: University of Cincinnati / OhioLINK 2014
Subjects:
Cellular Biology
chromosome breakage
Pot2
telomere
Tetrahymena
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=ucin1415283984
id ndltd-OhioLink-oai-etd.ohiolink.edu-ucin1415283984
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Cellular Biology
chromosome breakage
Pot2
telomere
Tetrahymena
spellingShingle Cellular Biology
chromosome breakage
Pot2
telomere
Tetrahymena
Cranert, Stacey
The functional evolution of telomere proteins in Tetrahymena thermophila
author Cranert, Stacey
author_facet Cranert, Stacey
author_sort Cranert, Stacey
title The functional evolution of telomere proteins in Tetrahymena thermophila
title_short The functional evolution of telomere proteins in Tetrahymena thermophila
title_full The functional evolution of telomere proteins in Tetrahymena thermophila
title_fullStr The functional evolution of telomere proteins in Tetrahymena thermophila
title_full_unstemmed The functional evolution of telomere proteins in Tetrahymena thermophila
title_sort functional evolution of telomere proteins in tetrahymena thermophila
publisher University of Cincinnati / OhioLINK
publishDate 2014
url http://rave.ohiolink.edu/etdc/view?acc_num=ucin1415283984
work_keys_str_mv AT cranertstacey thefunctionalevolutionoftelomereproteinsintetrahymenathermophila
AT cranertstacey functionalevolutionoftelomereproteinsintetrahymenathermophila
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-ucin14152839842021-08-03T06:27:54Z The functional evolution of telomere proteins in Tetrahymena thermophila Cranert, Stacey Cellular Biology chromosome breakage Pot2 telomere Tetrahymena Telomeres are nucleo-protein complexes that contain DNA sequence complexed with specialized telomere binding proteins. The role of telomeres is to protect chromosome ends from being recognized as DNA damage and act as a buffer for DNA loss at the chromosome terminus. Telomeric DNA is a tandemly repeated sequence that consists of a double-stranded portion followed by a shorter, single-stranded overhang. Pot1 is a telomere binding protein that, in mammalian cells, binds the single stranded G-overhang of the telomere. It functions in telomere length regulation as well as in preventing the telomere from activating a cell cycle checkpoint. Tetrahymena thermophila have two POT homologs, Pot1 and Pot2. Pot1 is a functional homolog of the mammalian Pot1 in that it binds to the telomeric G-overhang and prevents it from activating a DNA damage response; however the function of Pot2 appears to be quite different. We have found that while Pot1 is essential during vegetative growth, Pot2 is not and Pot2 does not appear to have a role at macronuclear telomeres. RT-PCR and immunofluorescence analysis indicates that Pot2 is only expressed during the sexual phase of the Tetrahymena life cycle and it localizes to the developing macronucleus at a time when massive chromosome processing and new telomere formation occur. Chromatin immunoprecipitation (ChIP) assays demonstrate that Pot2 associates with sites of chromosome breakage (CBSs) but not sites of internal eliminated sequence (IES) excision during chromosome processing. In an effort to delineate the concurrent processes of chromosome breakage and IES excision, we analyzed the localization of the heterochromatic methylation marks, tri-methylated H3K9 and H3K27, during macronuclear development. It was known that these marks are essential for IES excision but their requirement for chromosome breakage was unclear. ChIP analysis shows that these marks are highly enriched at IESs but are not localized to CBSs. We also examined the requirement for expression of the essential IES excision protein Pdd1 in chromosome breakage. We confirm previous studies showing that parental expression of Pdd1 is not required for chromosome breakage, but demonstrate that complete loss of Pdd1 from both the parental and developing macronuclei results in ineffective chromosome breakage. Together, our results further support the evidence in the field that chromosome breakage and IES excision occur via different mechanisms but disruption of IES excision indirectly results in a defect in chromosome breakage. We have also begun the characterization of Pat2, a telomere protein that, unlike Pot2, functions at macronuclear telomeres in Tetrahymena. We show that knockout of Pat2 results in a telomerase-dependent decrease in telomere length without the rapid cell-cycle arrest seen with loss of Pot1. Co-immunoprecipitation analysis shows that Pat2 associates with the G-overhang binding complex in-vivo and this interaction is not dependent on DNA. These results indicate that Pat2 is a member of the G-overhang binding complex but it is unlikely to have a role in telomere protection. Instead Pat2 appears to positively regulate telomerase action at the telomere. The results presented in this dissertation demonstrate the evolution of two very different telomere protein homologs, Pot2 and Pat2. While Pat2 serves several canonical telomere protein functions, Pot2 has diverged to play other roles. Our analysis of Pot2 identifies the first protein in Tetrahymena to localize to CBSs and suggests that Pot2 could be involved in the exciting, and poorly characterized process of de novo telomere synthesis. 2014 English text University of Cincinnati / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ucin1415283984 http://rave.ohiolink.edu/etdc/view?acc_num=ucin1415283984 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

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