Ku is a heterodimeric protein involved in nonhomologous end-joining of the DNA double-stranded break repair pathway. It binds to the double-stranded DNA ends and then activates a series of repair enzymes that join the broken DNA. In addition to its function in DNA repair, the yeast Saccharomyces cerevisiae Ku (Yku) is also a component of telomere protein-DNA complexes that affect telomere function. The yeast telomeres are composed of duplex C(1-3)(A/T)G(1-3) telomeric DNA repeats plus single-stranded TG(1-3) telomeric DNA tails. Here we show that Yku is capable of binding to a tailed-duplex DNA formed by telomeric DNA that mimics the structure of telomeres. Addition of Cdc13p, a single-stranded telomeric DNA-binding protein, to the Yku-DNA complex enables the formation of a ternary complex with Cdc13p binding to the single-stranded tail of the DNA substrate. Because pre-loading of Cdc13p to the single-stranded telomeric tail inhibits the binding of Yku, the results suggested that loading of Yku and Cdc13p to telomeres is sequential. Through generating a double-stranded break near telomeric DNA sequences, we found that Ku protein appears to bind to the de novo synthesized telomeres earlier than that of Cdc13p in vivo. Thus, our results indicated that Yku interacts directly with telomeres and that sequential loading of Yku followed by Cdc13p to telomeres is required for both proteins to form a ternary complex on telomeres. Our results also offer a mechanism that the binding of Cdc13p to telomeres might prevent Yku from initiating DNA double-stranded break repair pathway on telomeres.
"Therefore, Ku may promote interaction between Cdc13 and Est1, leading to stable association of Est1 with the telomere. However, the binding of Ku to the telomeric end must occur prior to Cdc13 binding, as in vitro studies have shown that Ku cannot bind to an end prebound by Cdc13 (Wu et al. 2009). This suggests that Cdc13 would not yet be present at the telomere to receive telomerase handed off by Ku. "
[Show abstract][Hide abstract] ABSTRACT: Telomere length is tightly regulated in cells that express telomerase. The Saccharomyces cerevisiae Ku heterodimer, a DNA end-binding complex, positively regulates telomere length in a telomerase-dependent manner. Ku associates with the telomerase RNA subunit, TLC1, and this association is required for TLC1 nuclear retention. Ku-TLC1 interaction also impacts the cell cycle regulated association of the telomerase catalytic subunit Est2 to telomeres. The promotion of TLC1 nuclear localization and Est2 recruitment have been proposed to be the principal role of Ku in telomere length maintenance, but neither model has been directly tested. Here we study the impact of forced recruitment of Est2 to telomeres on telomere length in the absence of Ku's ability to bind TLC1 or DNA ends. We show that tethering Est2 to telomeres does not promote efficient telomere elongation in the absence of Ku-TLC1 interaction or DNA end binding. Moreover, restoration of TLC1 nuclear localization, even when combined with Est2 recruitment, does not bypass the role of Ku. In contrast, forced recruitment of Est1, which has roles in telomerase recruitment and activation, to telomeres promotes efficient and progressive telomere elongation in the absence of Ku-TLC1 interaction, Ku DNA end binding or Ku altogether. Ku associates with Est1 and Est2 in a TLC1-dependent manner and enhances Est1 recruitment to telomeres independently of Est2. Together, our results unexpectedly demonstrate that the principal role of Ku in telomere length maintenance is to promote the association of Est1 with telomeres, which may in turn allow for efficient recruitment and activation of the telomerase holoenzyme.
"Supporting this proposal are data showing that leading telomeres in vertebrates are particularly susceptible to end-joining reactions when Ku-associated factors are depleted (Bailey et al. 2001). Finally, since biochemical evidence suggests that Ku must slide internally to allow for CST binding (Fig. 1C; Wu et al. 2009), the accumulation of Ku-bound blunt-ended telomeres in Arabidopsis may reflect an early exit from the telomere cap assembly line. "
[Show abstract][Hide abstract] ABSTRACT: Telomeres ensure the complete replication of genetic material while simultaneously distinguishing the chromosome terminus from a double-strand break. A prevailing theme in telomere biology is that the two chromosome ends are symmetrical. Both terminate in a single-strand 3' extension, and the 3' extension is crucial for telomere end protection. In this issue of Genes & Development, Kazda and colleagues (pp. 1703-1713) challenge this paradigm using a series of elegant biochemical and genetic assays to demonstrate that half of the chromosomes in flowering plants are blunt-ended. This discovery reveals unanticipated complexity in telomeric DNA processing and a novel mode of chromosome end protection.
Genes & development 08/2012; 26(15):1648-52. DOI:10.1101/gad.199059.112 · 10.80 Impact Factor
[Show abstract][Hide abstract] 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.
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