Saccharomyces cerevisiae Tel2 plays roles in TORC signaling and telomere maintenance that can be mutationally separated.

The evolutionary conserved Tel2 protein appears to function as a co-chaperone required for the activity of phosphatidylinositol 3-kinase-like protein kinases (PIKKs). Since Saccharomyces cerevisiae Tel2 is essential for viability and only a single point mutant (Tel2-1) had been characterized so far, the possible range of phenotypes associated with Tel2 mutations was unknown. We used random in vitro mutagenesis and plasmid shuffling to create additional point mutants. No significant sensitivity towards DNA damaging agents or hydroxyurea was evident, indicating that Tel2 is not required for Mec1 function. However, as frequent novel phenotypes, we detected slow growth or enhanced lethality in response to rapamycin that could be correlated with lower level and activity of Tor1 or of both Tor1 and Tor2, respectively. The newly isolated mutant with the most severe phenotype, Tel2-13, is comprised of 8 amino acid changes. Two mutated residues of Tel2-13 near the N-terminus and close to Tel2-1 are sufficient for shortened telomeres whereas multiple mutations within the C-terminal two thirds of the protein are required for enhanced rapamycin lethality. Our findings demonstrate separation of function explainable by differential binding of Tel2 to its PIKK substrates Tel1 or Tor1/Tor2 and thus a critical contribution of Tel2 to the interface that links various PIKKs to this chaperone complex.

Regulation of Saccharomyces cerevisiae DNA polymerase eta transcript and protein.

RAD30-encoded DNA polymerase eta functions as a translesion polymerase that can bypass the most frequent types of UV-induced pyrimidine photoproducts in an error-free manner. Although its transcript is UV-inducible in Saccharomyces cerevisiae, Rad30 (studied as a Rad30-Myc fusion) is a stable protein whose levels do not fluctuate following UV treatment or during cell cycle progression. Rad30 protein is subject to monoubiquitination whose level is upregulated in G1 and downregulated during S-phase reentry. This downregulation is accelerated in UV-treated cells. A missense mutation (L577Q) of the ubiquitin binding domain (UBZ) confers a reduced degree of ubiquitination outside of G1 and a complete failure to stably interact with ubiquitinated substrates. This mutation confers a phenotype resembling a complete RAD30 deletion, thus attesting to the significance of the UBZ motif for polymerase eta function in vivo.