Materials and Methods

Strains and plasmids
The strains and plasmids used in this paper are described in Supporting Information, Table S1. Deletion and epitope-tagged strains were generated by one-step gene replacement or integration, respectively, with the noted selectable marker. All incubations were performed at 28°.

Telomere analysis
Telomere length analysis and determination of telomerase-independent survivors were performed as described previously (Lendvay et al. 1996).

Senescence progression assays
Serial liquid culture senescence progression assays were carried out as described previously (Le et al. 1999; Rizki and Lundblad 2001). To summarize, spore colonies from freshly dissected tetrads were inoculated in their entirety into YPD media and grown at 28° for 46 hr. Cell counts were determined via hemocytometer. Samples were diluted back into fresh media to a concentration of 1 × 105 cells/mL and grown for 22 hr at which time cells were counted. The cultures were again diluted back into fresh media to a concentration of 1 × 105 cells/mL. Cell counts and dilution into fresh media were repeated every 22 hr. Several isolates of each genotype were examined.

Growth experiments
To assess growth in the BIR assay strain, fivefold serial dilutions of exponentially growing liquid cultures in YPLactate were plated on YPD or YPGal. To assay strains containing plasmids, the cultures were pre-grown in -Leu, -Trp, or -Ura minimal media, as appropriate, to maintain selection of the plasmid. To assess growth under conditions of stress induction, cultures were pregrown in either YPD for the YPH strain or YPLactate for the BIR assay strain and plated for single colonies on YPD medium containing 2−4 µg/mL bleomycin, 0.25% glucose, 0.5 M NaCl, or 3 mM hydrogen peroxide or at 37°. To assess growth under conditions of added tryptophan, fivefold serial dilutions of liquid cultures grown in YPLactate were plated on YPD, YPD + 100 μM tryptophan, YPGal, and YPGal + 100 μM tryptophan. To assess growth with added quinolinic acid, fivefold serial dilutions of liquid cultures grown in YPLactate were plated on YPD, YPD + 2 μM or 4 μM quinolinic acid, YPGal, and YPGal + 2 μM or 4 μM quinolinic acid. All phenotypes were recorded 4 days postplating unless otherwise specified.

BIR assays
BIR plating and determination of percent viability were performed as described previously (Lydeard et al. 2007). Sensitivity to canavanine and/or hygromycin determined whether repair occurred via BIR or another pathway. All individual colonies on the YPGal plate were picked and inoculated into 96-well dishes. The cell suspensions were then pinned onto YPD, canavanine and hygromycin plates.

HO induction and repair kinetics
Strains were pregrown in YPLactate. Galactose induction, sample collection and processing, and DNA analysis were performed as described previously (Lydeard et al. 2010).

Protein analysis under galactose induction
Strains were pregrown in YPLactate to approximately 0.5 × 107 cells. Initial aliquots were taken and then galactose was added to each culture to a final concentration of 2%. Aliquots were taken at the indicated time points. Samples were spun at 3000 rpm and cell pellets washed twice with water before being frozen at −80°. The cell pellets were thawed and normalized to cell count before lysis. For analysis of Rad53 phosphorylation, protein lysates were prepared by trichloroacetic acid method as previously described (Foiani et al. 1994). For analysis of Tat2 protein levels, samples were prepared as previously described (Abe and Iida 2003). Before western analysis of Tat2, 50 µg of whole-cell extract was denatured in 5% SDS and 5% β-mercaptoethanol at 37° for 10 m. Western blots were probed with α-Flag (F3165; Sigma-Aldrich), α-Rad53 (provided by M. Foiani), and α-PGK (ab113687; Abcam) antibodies.