Functional Analysis of the Yku Complex in Telomere Length Regulation

Abstract

The Ku protein from Saccharomyces cerevisiae (Yku) forms, like its human homologue hKu, a heterodimer comprised of a 70kD and an 80 kD subunit. In yeast and mammals, the Ku heterodimer is required for the repair of DNA double strand breaks (DSBs) via nonhomologous end-joining (NHEJ). Interestingly, Ku has been shown to bind to the native chromosome ends. It contributes to the maintenance of wild type telomere length and, moreover, has been implicated in the protection of the telomeres from end-to-end fusions. Telomere-bound Yku delocalizes from telomeric foci in response to DNA damage and accumulates at the sites of a DNA break. This thesis aimed to further characterize the Yku heterodimer and its function at DSBs and the native chromosome ends. In a genetic screen for mutations that - in combination with a yku deletion - lead to cell death, a novel mutation in the yeast telomerase subunit CDC13/EST4 has been identified earlier in the laboratory. Cdc13p binds to the single stranded DNA overhang at telomeres and is required to recruit the yeast telomerase to chromosome ends. The results presented here suggest that the mutant protein, Cdc13-4p, can still bind to the telomere and does interact with the telomerase subunit Est1p in vivo. A model is proposed in which the mutant Cdc13p is altered in its binding to a regulatory protein, thereby modulating telomerase access to the chromosome ends. The lethal effect in yku mutants is discussed to result from the loss of additional telomere sequences at the already very short telomeres of yku mutants. In order to fulfill its opposite functions at the ‘different’ DNA ends, Yku might depend on larger protein networks. Putative Yku interacting proteins have been identified in a two hybrid screen. One interactor, Sir4p, has previously been implicated in NHEJ. The Sir4p domain identified could be shown to interact with the Yku heterodimer via the Yku80p subunit. Experiments that allowed the separation of phenotypes caused by the loss of the Sir4 protein itself and phenotypes induced by a de-repression of silencing in sir4 mutants revealed no direct involvement of Sir4p in the repair of DSBs. In contrast to Cdc13p, Sir4p acts epistatic with Yku at the telomeres, indicating that the protein-protein interaction detected by two hybrid criteria might take place at telomeres. Besides defects in DNA repair and telomere protection, mice deficient for Ku have been reported to exhibit phenotypes indicative of premature aging. Loss of yku70 or overexpression of the Yku heterodimer effects life span in yeast. Experiments presented here rise the possibility that the premature aging is correlated with Ku’s function at the telomere