Telomeres, the ends of eukaryotic chromosomes have emerged as being a crucial link in carcinogenesis and cellular senescence in humans. In the great majority of eukaryotes, telomeres are composed of tandem arrays of a short repeat (TTAGGG in humans) that serve as binding sites for proteins that protect the ends from degradation and fusion. Because telomeres cannot be fully replicated by normal DNA polymerases, some sequence can be lost from their ends each time a cell divides. This is averted in unicellular eukaryotes and human germ line cells because of the action of telomerase, a ribonucleoprotein enzyme that adds copies of the telomeric repeat de novo onto chromosome ends. Recent evidence has indicated that shortened telomeres may underlie the limited proliferative capacity of normal human somatic cells (which have little or no telomerase). Immortalized human cells, both laboratory cell lines and those derived from cancers, universally show a restoration in their ability to maintain telomeric repeats at their chromosome ends. While in most cases, this restoration is associated with restored telomerase activity, a significant minority have acquired the ability to maintain telomeres using a mechanism independent of telomerase, termed ALT, that is thought to involve recombination. This application is aimed primarily at understanding the molecular basis of recombinational telomere maintenance in the yeast Kluyveromyces lactis, which closely resembles ALT seen in human cells.
Specific aims of this proposal will be directed at a number of aspects of telomeric recombination. A novel silent telomere mutation will be used to measure for the first time recombination rates within functionally normal telomeric repeat arrays and to study the mechanism involved in the recombination that becomes specifically induced near shortened telomeres. The repetitive subtelomeric sequences of K. lactis will be characterized to help understand their role in recombinational telomere maintenance and to provide tools for other experiments. Most effort will be used to study recombinational telomere elongation in cells with little or no telomerase. Preliminary evidence indicates that recombinational telomere lengthening of K. lactis telomeres can occur very efficiently by utilizing a circular DNA molecule as a template. Experiments will be undertaken to examine whether rolling circle replication of small telomeric DNA circles may underlie the superficially very different forms of recombinational telomere lengthening seen in K. lactis and in Saccharomyces cerevisiae.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061645-04
Application #
6630461
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Carter, Anthony D
Project Start
2000-09-01
Project End
2005-08-31
Budget Start
2003-09-01
Budget End
2004-08-31
Support Year
4
Fiscal Year
2003
Total Cost
$224,440
Indirect Cost
Name
University of Georgia
Department
Genetics
Type
Schools of Arts and Sciences
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
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Xu, Jianing; McEachern, Michael J (2012) Maintenance of very long telomeres by recombination in the Kluyveromyces lactis stn1-M1 mutant involves extreme telomeric turnover, telomeric circles, and concerted telomeric amplification. Mol Cell Biol 32:2992-3008
Hsu, Min; Yu, Eun Young; Sprusansky, Ondrej et al. (2012) Functional analysis of the single Est1/Ebs1 homologue in Kluyveromyces lactis reveals roles in both telomere maintenance and rapamycin resistance. Eukaryot Cell 11:932-42
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Basenko, Evelina; Topcu, Zeki; McEachern, Michael J (2011) Recombination can either help maintain very short telomeres or generate longer telomeres in yeast cells with weak telomerase activity. Eukaryot Cell 10:1131-42
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Bechard, Laura H; Jamieson, Nathan; McEachern, Michael J (2011) Recombination can cause telomere elongations as well as truncations deep within telomeres in wild-type Kluyveromyces lactis cells. Eukaryot Cell 10:226-36
Basenko, Evelina Y; Cesare, Anthony J; Iyer, Shilpa et al. (2010) Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination. Nucleic Acids Res 38:182-9
Wang, Zhi-Ru; Guo, Leilei; Chen, Lizhen et al. (2009) Evidence for an additional base-pairing element between the telomeric repeat and the telomerase RNA template in Kluyveromyces lactis and other yeasts. Mol Cell Biol 29:5389-98
Cesare, Anthony J; Groff-Vindman, Cindy; Compton, Sarah A et al. (2008) Telomere loops and homologous recombination-dependent telomeric circles in a Kluyveromyces lactis telomere mutant strain. Mol Cell Biol 28:20-9

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