title: Ku must load directly onto the chromosome end in order to mediate its telomeric functions.
abstract: The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding-defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance.
authors.affiliation: Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America. abertuch@bcm.edu
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Lopez
authors.forename: Albert
authors.initials: A
authors.lastname: Ribes-Zamora
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Indiviglio
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Williams
authors.forename: Svasti
authors.initials: S
authors.lastname: Haricharan
authors.forename: Alison A
authors.initials: AA
authors.lastname: Bertuch
mesh_terms: D015825:Chromosomes, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D047468:Immunoprecipitation; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism; D016615:Telomere / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C061573:SIR4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; C081647:high affinity DNA-binding factor, S cerevisiae; D019098:Telomerase