Sequential Loading of Saccharomyces cerevisiae Ku and Cdc13p to Telomeres

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.DNA damages in the form of double-stranded breaks (DSBs)⁴ compromise the integrity of genomes. Failure in repairing or mis-repairing double-stranded breaks can lead to chromosome instability and eventually cell death or cancer (1). Double-stranded breaks are repaired by two main pathways, the homologous recombination and nonhomologous DNA end-joining. In nonhomologous DNA end-joining, Ku is the first protein to bind to the DNA ends to initiate the repair pathway (2). Upon binding, Ku then recruits a series of repair enzymes to join the broken ends (2). Ku is a heterodimeric protein composed of 70- and ∼80-kDa subunits. In Saccharomyces cerevisiae, Ku includes Yku70 and Yku80 subunits. Because the biochemical configuration of the broken ends could be very diverse on DSBs, Ku binds to double-stranded ends in a sequence- and energy-independent manner. It is capable of binding to DNA ends with blunt 3′-overhangs or 5′-overhangs as well as double-stranded DNA with nicks, gaps, or internal loops (3–7). However, Ku does not have high affinity to single-stranded DNA. The crystal structure of human Ku heterodimer indicates that it forms a ring structure that encircles duplex DNA (7). This unique structure feature enables Ku to recognize DNA ends and achieves its high affinity binding.In additional to the role in double-stranded break repair, Ku was shown to be a component of telomeric protein-DNA complex in yeast and mammals (8–10). Telomeres are terminal structures of chromosomes composed of short tandem repeated sequences (11, 12). Mutation of YKU70 or YKU80 causes defects in telomere structure (13–15), telomere silencing (16–19), and replication timing of telomeres (20). The function of yeast Ku (Yku) on telomeres could mediate through protein-protein interaction with Sir4p or protein-RNA interaction with Tlc1 RNA (21, 22). For example, through the interaction with Sir4p, Yku selectively affects telomeres silencing but not the silent mating type loci (17). Yku could also bind to telomerase Tlc1 RNA for telomere length maintenance (22). Judged by the DNA binding activity of Yku, it is reasonable to suggest that it may bind directly to telomeric DNA. Indeed, it was shown that human Ku is capable of binding directly to telomeric DNA in vitro (15). Moreover, because the deletion of SIR4 in budding yeast (23) or Taz1 in fission yeast (24) does not abolish the association of Ku with chromosomal ends, this suggests that Ku might bind directly to telomeric DNA in cells. However, because yeast telomeres have a short 12–14-mer single-stranded tail (25), it is uncertain whether Yku could pass the single-stranded region to reach its binding site. The direct binding of Yku to telomeric DNA has not been experimentally determined.In contrast to double-stranded breaks, the ends of linear chromosomes are not recognized by repair enzymes as DNA damage. In S. cerevisiae, Cdc13p is the single-stranded TG_1–3 DNA-binding protein that enables cells to differentiate whether the ends of a linear DNA are telomeres or broken ends (26–29). Thus, although the mechanism of how cells prevent the activation of DSB repair pathway in telomere is unclear, it is likely that binding of Cdc13p to telomeres might inhibit the initiation of DNA damage response by the Ku protein. Here, using a tailed-duplex DNA synthesized by telomeric DNA sequences to mimic telomere structure, we showed that Yku binds directly to this tailed-duplex DNA substrate and forms a ternary complex with Cdc13p. Our results also showed that Yku loaded to a de novo synthesized telomere earlier than Cdc13p in vivo. These results support the direct binding of Yku to telomeric DNA and that the spatial orientation of Cdc13p might block the activation of DSB repair pathway on telomeres.