In most eukaryotes, telomere length is maintained by telomerase, a ribonucleoprotein that adds TG-rich telomeric repeats de novo to chromosome ends. Telomeric binding proteins (for example, Rap1p) affect telomere length by negatively regulating the access of telomerase to the telomeres. However it has been unclear whether telomerase directly participates in this regulation. In S. cerevisiae, telomerase has at least four components, among which Est2p (Telomerase Reverse Transcriptase subunit) and TLC1 RNA compose the catalytic core. This thesis describes analysis of the mechanism through which several mutations identified in the N-terminus of Est2p (est2-LT) cause telomere over-elongation and uncovers a novel role of telomerase in influencing the association of Rap1p with telomeres.
est2-LT mutants behave normally in standard primer extension assays, and have normal telomerase composition and telomere end structure. These data suggest that the LT mutations might affect a regulatory function of telomerase without impairing its intrinsic enzymatic activity. Here I show that the association of Rap1p (per nucleotide) is reduced in est2-LT mutants. Telomere over-lengthening in est2-LT is dependent on downstream effectors of the Rap1p pathway (RIF1, RIF2 and TEL1), consistent with the hypothesis that the reduced binding of Rap1p in vivo allows increased access of telomerase to telomeres. This behavior contrasts with a mutation in EST2 (est2-up34) that increases the association of Rap1p as expected for a strain with long telomeres. Although I observe changes in the telomere sequences of est2-LT strains, est2-up34 strain shows the same alterations, suggesting that the sequence changes are a consequence, rather than a cause of telomere over-elongation. Consistent with this idea, there are no significant differences in the association of Rap1p with WT or est2-LT telomeres in vitro. Taken together, I propose that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, and that telomerase has an additional role upstream of Rap1p in telomere length homeostasis. A mutation within the same region of S. pombe TERT subunit causes telomere lengthening and alters telomeric sequences, suggesting that this function of TERT might be evolutionarily conserved.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-12032007-110635 |
| Date | 06 December 2007 |
| Creators | Ji, Hong |
| Contributors | Todd R. Graham, Katherine L. Friedman, Ellen Fanning, Christopher F. J. Hardy, James G. Patton |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-12032007-110635/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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