FIG. 2.
The ILT pathway of postsenescence survival amplifies telomeric TG1-3 repeats. (A) Representative Southern blots (XhoI digestion, TG1-3 probe) showing telomere organization in a tlc1Δ rad52Δ (rad52::LEU2 allele [top panel] or rad52::KanMX4 allele [bottom panel]) mutant obtained by sporulating a tlc1Δ/tlc1Δ rad52Δ/RAD52+ diploid already recombining in type II (following growth in liquid medium for ∼150 generations; see Fig. 1B, left panel). Cells were restreaked every 3 days on agar-based medium (YEPD, 29°C) for the indicated number of generations (around 30 generations per restreak under these conditions). The bulk size of wild-type telomeres, around 1.3 kb, is indicated by the arrowhead (lane 1). The three arrows at the bottom of each gel indicate three different times when critically short telomeres reelongated, indicated by the disappearance of the band with the lowest size at the next time point. Note that the slope of telomere shortening in the bottom panel is less than that in top panel because in the former, samples were taken every 20 generations, versus 30 generations in the latter. Ten experiments in total were performed with the rad52::LEU2 allele and eight with the rad52::KanMX4 allele. (B) Recutting samples 1 to 6 from the top panel in panel A with a mixture of restriction enzymes (AluI, HaeIII, HinfI, and MspI), using a 4-bp recognition sequence, which do not cut within the TG1-3 repeats (68; see also Materials and Methods) indicates that TG1-3 sequences are amplified in the Rad52-independent survivors. Rad52-dependent type I and II controls are shown in Fig. 4C. A TG1-3 probe was used. (C) Telomere organization in a tlc1Δ rad52::LEU2 strain (representative of a total of four experiments) which previously contained a RAD52-URA3 plasmid and was recombining in type II, after losing the RAD52 plasmid on 5-FOA (see Fig. 1B, middle panel). The same cells as those illustrated in Fig. 7B, first column, are shown. XhoI digestion and a TG1-3 probe were used.