Mechanisms underlying bypass of the essential telomere-capping protein Cdc13

Abstract

In S. cerevisiae, Cdc13-Stn1-Ten (CST) caps chromosome ends (telomeres). Cdc13 binds telomeric single-stranded DNA and with Stn1 and Ten1 protects the telomere end from attack by DNA damage response (DDR) factors. All three CST genes are essential for cell viability, however the requirement for CDC13 can be bypassed in strains lacking the nonsense-mediated mRNA decay (NMD) gene NMD2 and the DDR genes EXO1 (encoding a 3’-5’ exonuclease) and RAD24 (encoding a DNA damage checkpoint protein). This PhD aimed to determine the mechanisms underlying Cdc13 bypass. One mechanism identified was that Stn1 is at telomeres in the absence of Cdc13, suggesting that Stn1 has a telomere protection role separate from Cdc13. To uncover the mechanisms underlying Cdc13 bypass, a synthetic genetic array (SGA) screen was conducted using an nmd2 exo1 cdc13 query strain crossed with a library of single gene deletion mutants. Rif1, which has telomeric and genome-wide roles, was found to be an absolute requirement for Cdc13 bypass. This result was confirmed by low-throughput methods. By analysing the phenotypes of rif1 mutant alleles in a viable cdc13 strain it was possible to deduce the functions of Rif1 required for Cdc13 bypass. Remarkably, the putative HEAT-repeat domain and Glc7 (Protein Phosphatase 1) interaction sites were found to be essential. It is proposed that Rif1 uses this domain to interact with Ten1 to link CST with Rif1/Rap1/Rif2 to form a shelterin-like complex. Finally, evidence was found that Rad52-mediated recombination and long telomeres, with extended TG1-3 repeats, promote Cdc13 bypass. Overall, multiple mechanisms underlie Cdc13 bypass: Stn1 activity, Rif1 interactions and recombination of telomeric TG1-3 repeats.