Genetic requirements of Rad52-dependent and Rad52-independent postsenescence survival pathways

Relevant genotypeaSurvivor typebComments
tlc1I or II-ALTTelomeres continuously shorten, as previously reported (68); after 700-800 cell divisions, telomeres convert from type II to type I most of the time (Fig. 9A)
tlc1 + TLC1Telomerase controlNormal telomere length regulation restored when TLC1 is reintroduced in type II survivors (Fig. 9C)
tlc1 rad50IRad59 essential for type II-ALT
tlc1 rad51II-ALTRad51 essential for type I
tlc1 rad52 (-7::LEU2 or ::KAN)II-ILTEvidence for existence of the ILT pathway (Fig. 2A)
tlc1 rad59IRad50 essential for type II-ALT
tlc1 rfa1-t11II-ALTRfa1 essential for type I
tlc1 rad50 rad51II-ALTRad50 dispensable for type II-ALT when Rad51 is absent
tlc1 rad50 rad52-7::LEU2— (6)Rad50 essential for ILT
tlc1 rad50 rad52::KAN— (3)Rad50 essential for ILT
tlc1 rad50 rad59IRad50 and Rad59 essential for type II-ALT
tlc1 rad50 rfa1-t11 (6-15)Rfa1 essential for type I
tlc1 rad51 rad52 (-7::LEU2 or ::KAN)II-ILTRad51 dispensable for ILT
tlc1 rad51 rfal-tl1II-ALTRfa1 dispensable for type II-ALT
tlc1 rad59 rad52-7::LEUII-ILTRad59 dispensable for ILT
tlc1 rfa1-t11 rad52::KANcII-ILTRfa1 dispensable for ILT
tlc1 rad59 rfa1-tl1 (2)Rfa1 essential for type I
tlc1 rad50 rad51 rad52-7::LEU2 (6)Rad50 essential for ILT
tlc1 rad50 rad51 rad59 (15)No possible survival in the absence of Rad50, Rad51, and Rad59
tlc1 rad50 rad51 rfa1-t11— (15)Rfa1 essential for type II-ALT in the absence of Rad50
tlc1 rad50 rad59 rfa1-t11— (2)Rfa1 essential for type I
tlc1 rad52::KAN
    elg1d— (7)
    rad1d— (8)
    mre11d— (6)
    rad50d— (3)
tlc1 rad52-7::LEU2
    rad50d— (6)
    rad50 rad51d— (6)
tlc1 rad52::KAN
  • a A “young” tlc1Δ RAD52+ mutant (prior to the onset of senescence) was mated to a “young” tlc1Δ mutant harboring one or several mutations in the indicated gene(s). The resulting diploid strain was then allowed to recombine during growth in liquid cultures for ∼150 generations, which yielded exclusively type II survivors. After sporulation, mutants of the desired genotype were selected and grown on agar-based plates. The cultures were then propagated by restreaking every 3 days (∼30 cell divisions per passage, at 29°C) for 90 to 130 days.

  • b Survivor type (I or II) was determined after visualizing telomere structure (see Fig. 1C) by Southern blotting (see Materials and Methods). The samples were prepared after 90 days of growth on plates (following selection of the mutant as explained in footnote a), which corresponds to ∼900 cell divisions. In some cases, when phenotype confirmation was needed, cultures were further propagated for 14 additional passages. —, cell death at the passage indicated in parentheses.

  • c Opposite results were obtained with the rad52-7::LEU2 background (see text).

  • d Essential for ILT.

  • e Dispensable for ILT.