Maintenance of Double-Stranded Telomeric Repeats as the Critical Determinant for Cell Viability in Yeast Cells Lacking Ku

Abstract

The Saccharomyces cerevisiae Ku complex, while important for nonhomologous DNA end joining, is also necessary for maintaining wild-type telomere length and a normal chromosomal DNA end structure. Yeast cells lacking Ku can grow at 23°C but are unable to do so at elevated temperatures due to an activation of DNA damage checkpoints. To gain insights into the mechanisms affected by temperature in such strains, we isolated and characterized a new allele of the YKU70 gene, yku70-30^ts . By several criteria, the Yku70-30p protein is functional at 23°C and nonfunctional at 37°C. The analyses of telomeric repeat maintenance as well as the terminal DNA end structure in strains harboring this allele alone or in strains with a combination of other mutations affecting telomere maintenance show that the altered DNA end structure in yeast cells lacking Ku is not generated in a telomerase-dependent fashion. Moreover, the single-stranded G-rich DNA on such telomeres is not detected by DNA damage checkpoints to arrest cell growth, provided that there are sufficient double-stranded telomeric repeats present. The results also demonstrate that mutations in genes negatively affecting G-strand synthesis (e.g., RIF1) or C-strand synthesis (e.g., the DNA polymerase α gene) allow for the maintenance of longer telomeric repeat tracts in cells lacking Ku. Finally, extending telomeric repeat tracts in such cells at least temporarily suppresses checkpoint activation and growth defects at higher temperatures. Thus, we hypothesize that an aspect of the coordinated synthesis of double-stranded telomeric repeats is sensitive to elevated temperatures.

We thank D. Gottschling, S. Elledge, J. Haber, S.-E. Lee, S. Labbé, and V. Lundblad for providing yeast strains and plasmids. We also thank Stéphanie Larose for her help with RNA preparations. Members of the Wellinger lab are thanked for their continued intellectual input, and K. Runge is thanked for his insightful comments on the manuscript.

This work was supported by CCS research grant 010049 from the National Cancer Institute of Canada (NCIC) and a core facility group grant by the Canadian Institutes of Health Research (MGC-48372). R.J.W. is a Chercheur-National of the FRSQ.