The role of novel and known telomeric chromatin‐associated proteins in suppression of DNA instability in Ku mutants of S. cerevisiae

Abstract

The Yku70/Yku80 protein heterodimer is essential for precise rejoining of the ends of broken chromosomes after cells are exposed to DNA damaging agents such as X-rays or strand-breaking chemicals. The complex is also a component of the specialized protein structures normally found at chromosome ends, or telomeres, and is believed to serve a protective function there. Yeast cells (Saccharomyces cerevisiae) with either the YKU70 or YKU80 gene inactivated are viable, but have unstable chromosome ends. This instability is exacerbated at slightly elevated growth temperatures and cells arrest growth in G2 phase of the cell cycle and ultimately lose viability. Interestingly, high temperature killing of yku70 mutants is prevented when intracellular levels of specific subunits of the telomerase DNA replication complex are increased. For example, overexpression of TLC1, the RNA subunit of telomerase, or EST2, which encodes the polymerase subunit, have both been found to prevent death of yku70 and yku80 mutants. The mechanism of this rescue is unknown. My research project involves an analysis of the genes and mechanism(s) involved using two approaches. The project will begin by investigating the role of telomeric chromatin proteins Sir3, Sir4, Rif1, and Rif2 in yku70 mutant rescue. The expression of these proteins will be elevated and the rescue effect at high temperature measured. Next, novel genes involved in Ku mutant rescue at high temperature will be explored using a library screen. A gene library, which uses a regulatory GAL1 promoter to express yeast cDNAs at high levels will be used. These two approaches will lead to a better understanding of the mechanism of telomere stabilization by telomeric chromatin proteins and the identification of new genes involved in telomere stability.