FIG. 9.
Evolution of postsenescence survival type with time in tlc1Δ RAD52+ cells. (A) In telomerase-negative mutants grown on solid (agar-based) medium, telomeres shortened progressively with passages (cells were restreaked, at 29°C, on agar-based plates every 3 days for the indicated number of generations). These survivors still exhibited a type II pattern until they attained their maximal shortening (left panel). At this point, telomeres in most of these old tlc1Δ mutants converted to a type I pattern (right panel), as seen in a total of 12 experiments. Indeed, the presence of an intense band at ∼0.9 to 1.0 kb following XhoI digestion, as well as the absence of any fragment above that size, suggested amplification of Y′ subtelomeric sequences (see Materials and Methods). In the remaining three cases, one or two additional bands remained visible over the ∼1.0-kb band (not shown). (B) When tlc1Δ mutants were propagated in liquid cultures, recombination on the telomeric TG1-3 sequences, indicated by the reappearance of numerous high-molecular-weight bands following XhoI digestion, persisted even after very long periods of time, as also seen in three additional experiments. The bulk size of wild-type telomeres, around 1.3 kb, is indicated by the arrowhead (lanes wt). A TG1-3 probe was used for panels A and B. (C) Pattern of Rad52-dependent telomeric recombination in a tlc1Δ mutant expressing TLC1 on a centromeric plasmid, introduced in the already-recombining tlc1Δ/tlc1Δ diploid. Progressively, the telomeric pattern varies from one typical of ongoing recombination (at the time of TLC1 introduction) to one indicating telomerase-based telomere length homeostasis, with, however, telomeres eventually stabilizing at a shorter-than-wild-type size, indicated by the arrowhead (wt, lane 1). Only one such experiment was performed. XhoI digestion and a TG1-3 probe were used.