The three-way junction is crucial for K. lactis telomerase function in vivo. The mutations introduced into the TWJ (A and B) and the resulting mean telomere length, as measured by Southern analysis (C), are summarized in (A). WT and mutant TER1 genes were introduced into K. lactis cells on a CEN-ARS plasmid, replacing the WT TER1 gene. These mutants contain an additional BclI template mutation that is incorporated into telomeres, introducing a BclI restriction site. Otherwise, the BclI mutation is phenotypically silent and can therefore be used to mark the nascent products of the investigated telomerase in vivo (see scheme in Figure S4). Genomic DNA was prepared from the sixth passage (∼90–120 generations), digested with EcoRI or EcoRI+BclI, electrophoresed on a 1% agarose gel, vacuum blotted onto a membrane, and hybridized first with a BclI-specific telomere probe (top) and then with a WT telomeric probe (bottom), as indicated on the right of (C). For simplicity, only a portion of the gel is shown, including 7 of the 12 K. lactis telomeres. (D) Typical colonies of the different strains taken at their forth passage. Impaired telomere maintenance is associated with rough colonies, as opposed to the smooth appearance of WT colonies.