The Ku Heterodimer Performs Separable Activities at Double-Strand Breaks and Chromosome Termini

Abstract

The Ku heterodimer functions at two kinds of DNA ends: telomeres and double-strand breaks. The role that Ku plays at these two classes of termini must be distinct, because Ku is required for accurate and efficient joining of double-strand breaks while similar DNA repair events are normally prohibited at chromosome ends. Toward defining these functional differences, we have identified eight mutations in the large subunit of the Saccharomyces cerevisiae Ku heterodimer (YKU80) which retain the ability to repair double-strand breaks but are severely impaired for chromosome end protection. Detailed characterization of these mutations, referred to as yku80^tel alleles, has revealed that Ku performs functionally distinct activities at subtelomeric chromatin versus the end of the chromosome, and these activities are separable from Ku's role in telomere length regulation. While at the chromosome terminus, we propose that Ku participates in two different activities: it facilitates telomerase-mediated G-strand synthesis, thereby contributing to telomere length regulation, and it separately protects against resection of the C-strand, thereby contributing to protection of chromosome termini. Furthermore, we propose that the Ku heterodimer performs discrete sets of functions at chromosome termini and at duplex subtelomeric chromatin, via separate interactions with these two locations. Based on homology modeling with the human Ku structure, five of the yku80^tel alleles mutate residues that are conserved between the yeast and human Ku80 proteins, suggesting that these mutations probe activities that are shared between yeast and humans.

ACKNOWLEDGMENTS

We thank Nathan Walcott, Monika Walterscheid, Priya Kayath, Pinaki Patel, Olive Botor, and Amy Aroopala for technical assistance; Kara Nyberg and Ted Weinert for sharing their G-tail protocol; and Jeff Bachant, Jim Haber, and Dan Gottschling for generously providing strains and plasmids. Special thanks go to Deborah Wuttke for comments on the Ku80 protein alignment and Srinivasan Madubushi for assistance with rendering the Ku structure. We also thank members of the Lundblad lab for helpful discussions and Rachel Cervantes, Jim Huang, and Deborah Wuttke for critical reading of the manuscript.

This work was supported by a Baylor College of Medicine Child Health Research Center New Project Development Award (to A.B.), NIH grant K08 HD01231 (to A.B.), and NIH grant R01 AG16626 (to V.L.).