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.

Interplay between Ku and Replication Protein A in the Restriction of Exo1-mediated DNA Break End Resection

“…1A) (41). Consistent with published results, Ku-RE was found to be partially defective in binding blunt dsDNA ends when analyzed by an EMSA (Fig.…”

Chromosome end protection by blunt-ended telomeres

“…Resection is almost completely inhibited in angiosperm plants, resulting in telomeres with no or only a few-nucleotide long G-overhangs. Studies in budding yeast demonstrated that Ku physically associates with and protects chromosome termini in nondividing cells (Bonetti et al 2010;Vodenicharov et al 2010) and that telomeric function requires direct loading of Ku onto DNA through a circular channel in its structure (Lopez et al 2011;Pfingsten et al 2012). Blunt-ended telomeres generated by the leading strand replication are perfect substrates for Ku loading, which may be occluded after resection by proteins that bind to ssDNA (Faure et al 2010).…”

Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA

“…At least three different complexes contribute to the capping function and prevent telomeres from being recognized as DSBs: Rap1-Rif1-Rif2 bound to double-stranded telomeric repeats 7,8 , the CST (Cdc13, Stn1, Ten1) complex that covers the G-tail [9][10][11] and the Ku heterodimer that directly binds to the terminal telomeric DNA 12,13 . One role of these complexes is to control the action of the nucleases/helicases involved in DSB resection 14 .…”