Telomeres, the ends of eukaryotic chromosomes, are a crucial link in understanding carcinogenesis and cellular sensescence in humans. 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, some sequence can be lost from their ends each time a cell divides. This is normally averted by telomerase, a ribonucleoprotein enzyme that adds copies of the telomeric repeat onto chromosome ends. Much evidence indicates that telomere shortening underlies the limited proliferative capacity of normal human somatic cells (which have little or no telomerase). Immortal human cancers show a restored ability to maintain telomeres. In most cases, this restoration is associated with restored telomerase activity, but in some cases involve an alternative, recombination-independent form of telomere maintenance called ALT. In this application, a powerful yeast system, Kluyveromyces lactis, is utilized to understand how recombinational telomere elongation (RTE) occurs. Recent work has suggested that the RTE that happens in K. lactis mutants lacking telomerase occurs through a 'roll and spread' mechanism whereby a tiny circle of telomeric DNA (t-circle) is first copied by a rolling circle mechanism to make the first elongated telomere, followed by other telomeres copying sequence from that telomere. This proposal aims to do further experiments to characterize how RTE occurs, including an alternate type of RTE which produces extreme telomere elongation especially similar to what occurs in human ALT cells. Other experiments will be done to determine precisely which features permit a telomere to be 'capped' and lengthened by telomerase and which cause uncapping and trigger repair through recombination or non- homologous end joining. Still other work will test the hypothesis that t-circles are derived from a strand- invaded telomeric end (t-loop) that undergoes an intramolecular deletion event and test whether dysfunctional telomeres perturb recombinational repair throughout the genome. ? ? ?
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