Budding yeast Cdc13p is essential and a key protein involved in telomere maintenance and genome stability. Genetic and high-resolution structural studies of several of its fragments reveal an architectural organisation with five distinct domains , which include four OB-fold domains and one recruitment domain. However, many details on the in vivo roles of those domains and their potential inter-domain interactions remained elusive. In this work we constructed tagged versions of the Cdc13 protein and then generated five alleles, where each one contains a single deletion of a proposed domain. To study the roles of each domain in telomere maintenance, we took advantage of cdc13-independent cells. Although viable, these strains “Adapt” and inactivate check-point signalling to bypass the accumulation of telomeric DNA damage. The results show:
1- The first 80 amino acids of the OB1 domain contribute to stabilization of the Cdc13 protein. cdc13Δ-cells with this mutated protein show a strong capping defect. However this capping defect can be rescued when this mutated protein is co-expressed with the hypomorphic cdc13-1 allele. This result suggests a physical interaction and therefore supports a model where Cdc13 occurs as homodimer at the telomere. 2- Co-IP experiments, using two tagged-versions of Cdc13 strongly argue for this homodimer model. Surprisingly, the hypomorphic cdc13-1 allele doesn’t display any sign of dimerization defect at restrictive temperatures. Our data rather support the idea that Cdc13 dimerization requires at least two interfaces where the DBD domain contains the core dimerization interface. 3- The third domain (OB2) is essential for cell viability. cdc13-ob2Δ is unable to bind telomeric DNA in vivo and results in a capping deficiency in both cdc13Δ and cdc13-1 (ts) strains. This mutated protein still behaves as a dimer, indicating that OB2 is not involved in dimer formation. However, immunofluorescence analysis shows a mis-localized protein residing in the cytoplasm. Re-inserting a heterologous NLS peptide in place of OB2 restores nuclear localization, cdc13-independent capping and suppresses the ts phenotype of cdc13-1 mutant strain. Moreover, expression of the cdc13-ob2Δ-NLS results in hyper elongated telomere, suggesting an additional function. 4- Cdc13-independent cells are sensitive to damage induced by genotoxic drugs. Remarkably, ectopic expression of Cdc13 cells restores a normal resistance to those drugs. This observation suggests an unexpected role of Cdc13 in DNA damage response at telomeres. Results obtained with the same deletions as described above, we conclude that this sensitivity to genotoxic drugs in the cdc13Δ cells may be due to the eroded state of the telomeres in these cells.