FIG. 8.
Kinetics of telomeric recombination in RAD52+ telomerase-negative (tlc1Δ) cells in the absence of Rad50 (A), of Rad59 (B), of both Rad50 and Rad59 (C), or of both Rad50 and Rad51 (D). Data obtained with the tlc1Δ rfa1-t11 strain (E) showed that the essentiality of RPA in telomeric recombination (26) could be relieved by overelongated telomeres. Rad50 and Rad59 are individually essential for type II-ALT recombination (A and B) (both strains end up with a type I pattern), but in the absence of Rad51, Rad50 becomes dispensable for type II-ALT recombination, presumably carried out by Rad59 (D). The tlc1Δ rad50Δ rad51Δ rad59Δ (F) and tlc1Δ rad50Δ rad51Δ rfa1-t11 (G) mutants died at the 8th and 15th passage, respectively. Note that all seven strains were RAD52+. In all seven representative examples shown here, tlc1Δ mutants were issued from a type II-ALT recombining diploid homozygous for tlc1Δ and also bearing one or several additional null, heterozygous mutations in the RAD50, RAD51, or RAD59 gene. After sporulation of the obtained diploid, mutants of the desired genotype were restreaked on agar-based plates every 3 days for the indicated number of generations (estimating that at the temperature of 29°C, ∼30 generations were produced every 3 days on plates). The intense band at 1.2 to 1.3 kb in each first lane (wt), indicated by an arrowhead, represents the average size of the bulk of wild-type telomeres. Digestion of the genomic DNA preparations with XhoI followed by Southern blotting with a TG1-3 probe allowed the two types of recombination to be distinguished. Type I recombination (on the Y′ subtelomeric regions) yielded an XhoI-restricted terminal fragment around 0.9 to 1.0 kb, while type II survivors amplifying the TG1-3 repeats exhibited many XhoI fragments of different sizes, as explained in Materials and Methods. For each of the mutants illustrated in panels A to E and G, four experiments were performed, while three experiments were done for the mutant shown in panel F.