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.
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