Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends

Abstract

Ku is a conserved DNA end-binding protein that plays various roles at different kinds of DNA ends. At telomeres, Ku is part of the structure that protects the chromosome end, whereas at broken DNA ends, Ku promotes DNA repair as part of the nonhomologous end-joining (NHEJ) pathway. Here, we present evidence of a new role for Ku that impacts both telomere-length maintenance and DNA repair in Saccharomyces cerevisiae. We show that Ku binds TLC1, the RNA component of telomerase. We also describe a novel separation-of-function allele of Ku that is specifically defective in TLC1 binding. In this mutant, telomeres are short and the kinetics of telomere addition are slow, but other Ku-dependent activities, such as chromosome end protection and NHEJ, are unaffected. At low frequency, yeast will use telomerase to heal DNA damage by capping the broken chromosome with telomeric DNA sequences. We show that when Ku's ability to bind TLC1 is disrupted, DNA repair via telomere healing is reduced 10-to 100-fold, and the spectrum of sequences that can acquire a telomere changes. Thus, the interaction between Ku and TLC1 RNA enables telomerase to act at both broken and normal chromosome ends.

Opening the DNA repair toolbox: Localization of DNA double strand breaks to the nuclear periphery

“…14 A second pathway for telomerase recruitment also exists which involves interactions between TLC1, Est1/Est2/Est3 and the Ku70/80 heterodimer. 15,16 This latter mechanism study from Kramer and Haber demonstrated that de novo telomere formation is an extremely rare event at a persistent, HO-induced DSB. 41 In contrast, de novo telomere formation becomes efficient if a tract of 81 telomeric TG [1][2][3] repeats is inserted adjacent to the HO recognition site.…”

“…28 appears to play a key role in de novo telomere formation at DSBs that lack extensive TG repeats. 16,17 Persistent DSBs are Targeted to the Nuclear Periphery How does the cell respond when a DSB remains un-repaired for a long period of time? In yeast, cells can eventually inactivate the cell cycle checkpoint response (a process termed checkpoint adaptation) and proceed through mitosis with a broken chromosome, but adaptation occurs only after ∼15 hours of failed DSB repair.…”

“…16,40 Ku70 interacts directly with the TLC1 component of the telomerase complex, and inactivation of Ku70 has a dramatic effect on the efficiency of de novo telomere addition. 16 Furthermore, Shore and colleagues have demonstrated that Cdc13 and Ku70/ Ku80 cooperate in the recruitment of Est2 to an HO-induced DSB.…”

“…16,40 Ku70 interacts directly with the TLC1 component of the telomerase complex, and inactivation of Ku70 has a dramatic effect on the efficiency of de novo telomere addition. 16 Furthermore, Shore and colleagues have demonstrated that Cdc13 and Ku70/ Ku80 cooperate in the recruitment of Est2 to an HO-induced DSB. 14 To test the possibility that Ku70-dependent recruitment of the telomerase machinery may also contribute to peripheral localization of a persistent DSB, we monitored recruitment of Mps3 to a persistent DSB in isogenic wildtype and ku70Δ strains (Fig.…”

Actions of human telomerase beyond telomeres

“…It has been demonstrated that telomerase is transiently expressed in S-phase in some normal human cells, yet fails to maintain overall telomere length, but protect karyotypic stability by "capping"chromosome ends and by resetting chromatin during DNA replication [42] without adding telomeric repeats. An earlier report indicated that intrachromosomal DNA breaks are occasionally repaired by healing with telomeric DNA repeat sequences [43], suggesting that telomerase may participate in DNA repair. In addition, hTERT has been found to be associated with DNA replication protein primase [44].…”