FIG. 4.
Telomere amplification can take place in the simultaneous absence of RAD51 and RAD52 (A) or of RAD59 and RAD52 (B) and is of type II in both cases, as attested here by the band pattern generated following XhoI digestion and use of a TG1-3 probe. Cells were restreaked on agar-based plates every 3 days for the indicated number of generations. Both strains originated from a diploid already recombining in type II. The bulk size of wild-type telomeres, around 1.3 kb, is indicated by the arrowhead (lanes 1). Totals of four, three, and four experiments were performed for the strains illustrated in panel A left, panel A right, and panel B, respectively. (C) Confirmation of the type II nature of telomeric recombination in various telomerase-negative (tlc1Δ) mutants. For this, genomic DNAs previously analyzed using XhoI cutting were recut 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. Unlike these type II survivors, in which cutting with these enzymes did not eliminate the presence of numerous telomeric bands of various high molecular weights, the type I survivors of the tlc1Δ rad50Δ (lanes 6 and 15), tlc1Δ rad59Δ (lanes 9 and 18), and tlc1Δ rad50Δ rad59Δ (lanes 12 and 21) strains did not exhibit fragments higher than the wild-type telomere bulk size, around 1.2 kb. All strains were isolated from already-recombining (in type II) tlc1Δ/tlc1Δ diploids. All rad52Δ mutants shown here were of the rad52::LEU2 background. DNA from a nonrecombining wild-type strain is shown in lane 4. A TG1-3 probe was used.