FIG. 3.

Growth characteristics of the ILT survivors compared with the Rad52-dependent type I and II-ALT survivors. (A) Cells of the indicated relevant genotype were grown in liquid cultures (YEPD, 29°C). The ILT survivors were obtained by sporulating an already-recombining tlc1Δ/tlc1Δ RAD52⁺/rad52::KanMX4 diploid that had grown for ∼150 generations in liquid culture to generate type II-ALT survivors (not shown). Type I survivors were stabilized as such in liquid culture because of the introduced RAD51 deletion. Cells of these three haploid mutants were counted and diluted every day to 10⁵ cells/ml, and in parallel, 5 μl of each culture was dropped on agar medium, allowed to grow for 2 days, and photographed. (B) Another time course experiment, performed as for panel A, comparing in addition the rad52::KanMX4 and rad52::LEU2 strains. (C) Growth competition experiments between type I, type II-ALT, and type II-ILT survivors. Strains of the indicated relevant genotype (I and II refer to type I and type II survivors, respectively) were cultivated either separately (bottom three rows of cells) or as a mixture of two strains in the same flask (top two rows of cells) for 15 days, with dilution every day, in rich medium (YEPD) at 29°C (∼225 generations), and at intervals their growth was compared after spotting on selective media. To identify the strains having grown in the same culture, we took advantage of the nature of the auxotrophy conferred by the marker genes inserted at the disrupted loci, rad50::URA3, rad51::KanMX4, and rad52::LEU2. The ILT survivors (tlc1Δ rad52::LEU2) were rapidly outgrown by the Rad52-dependent type II survivors (tlc1Δ rad51::KanMX4) and competition stopped at day 7. On the other hand, the ILT and type I survivors were still competing after 11 days in culture, but the former then declined and disappeared. Southern analyses conducted in parallel established that these survivors were of the expected type of recombination (not shown). Only one experiment for each group of mutants (in panels A, B, and C) was performed.