Telomerase, required for the replication of chromosome ends in most eukaryotes, is unusual in that it is comprised of RNA as well as protein subunits. The goal of this project is to better understand structure- function relationships in yeast and human telomerases, with special attention to the regulation of telomerase action via the accessibility of its telomeric substrate. Understanding the fundamentals of telomerase action is important for human health, as 85 - 90% of cancers involve reactivation of telomerase; thus, there is substantial interest in anti-telomerase chemotherapeutic agents and in TERT (telomerase reverse transcriptase) as an antigen for cancer vaccines. In studies of yeast telomerase, we have proposed that the RNA subunit provides a flexible scaffold to tether essential proteins into the complex.
In specific aim #1, we will critically test this model in vitro and in vivo, and determine the structure and function of the TERT (Est2p)-binding region of the RNA.
In specific aim #2, we will analyze structure-function relationships of the yeast regulatory subunit Est1p and determine its functional interactions. We will test the specific hypothesis that the N-terminal region of Est1p is a 14-3-3 phosphoaminoacid-binding adapter involved in recruiting and activating telomerase at the telomere.
Specific aim #3 extends this work to the human system, taking advantage of our recent crystal structure of the chromosome-capping protein hPOT1 bound to telomeric single-stranded DNA. We will test the hypothesis that hPOT1 is switched from telomerase-inhibitory to faciliatory binding modes in a sort of """"""""chromatin remodeling"""""""" reaction, driven by post-translational modification and/or interaction with human EST1 proteins. ? ? ?
|Nandakumar, Jayakrishnan; Cech, Thomas R (2012) DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe. Nucleic Acids Res 40:235-44|
|Taylor, Derek J; Podell, Elaine R; Taatjes, Dylan J et al. (2011) Multiple POT1-TPP1 proteins coat and compact long telomeric single-stranded DNA. J Mol Biol 410:10-7|
|Zappulla, David C; Goodrich, Karen J; Arthur, Julian R et al. (2011) Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP. RNA 17:298-311|
|Latrick, Chrysa M; Cech, Thomas R (2010) POT1-TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation. EMBO J 29:924-33|
|Qiao, Feng; Goodrich, Karen J; Cech, Thomas R (2010) Engineering cis-telomerase RNAs that add telomeric repeats to themselves. Proc Natl Acad Sci U S A 107:4914-8|
|Zappulla, David C; Roberts, Jennifer N; Goodrich, Karen J et al. (2009) Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p. Nucleic Acids Res 37:354-67|
|Subramanian, Lakxmi; Moser, Bettina A; Nakamura, Toru M (2008) Recombination-based telomere maintenance is dependent on Tel1-MRN and Rap1 and inhibited by telomerase, Taz1, and Ku in fission yeast. Mol Cell Biol 28:1443-55|
|Qiao, Feng; Cech, Thomas R (2008) Triple-helix structure in telomerase RNA contributes to catalysis. Nat Struct Mol Biol 15:634-40|
|Mandell, Jeffrey G; Goodrich, Karen J; Bahler, Jurg et al. (2005) Expression of a RecQ helicase homolog affects progression through crisis in fission yeast lacking telomerase. J Biol Chem 280:5249-57|
|Zappulla, David C; Goodrich, Karen; Cech, Thomas R (2005) A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro. Nat Struct Mol Biol 12:1072-7|
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