Fig. 2.

The est2-LT^E76K mutation alters the alignment of telomerase with a short telomeric primer. (A) Illustration of the de novo telomere addition system developed by Diede and Gottschling (Diede and Gottschling, 1999). The left arm of chromosome VII is modified by addition (from centromere to telomere) of an ADE2 gene, 81 base pairs of telomere sequence, an HO endonuclease cleavage site and the LYS2 gene. Upon addition of galactose, expression of the HO endonuclease is induced and cleavage exposes a single-stranded overhang. De novo telomeres are isolated by ligation-mediated PCR using primers specific to the ligated oligonucleotide and to the ADE2 gene (bold). An example telomere synthesized by est2-LT^E76K telomerase is shown. The 5′-TG-3′ sequence assumed to facilitate primer–template alignment is italicized and the remainder of the HO endonuclease recognition site is highlighted in gray. The first core motif of the de novo telomere is underlined. (B) Time course of de novo telomere addition by EST2 (lanes 1–4) and est2-LT^E76K (lanes 5–8) telomerase. Cells were harvested immediately before HO induction (0 hours) or 1, 2 and 4 hours after galactose addition, as indicated. Marker sizes are in kilobases (lane M). Positions of the uncut product (no HO endonuclease cleavage) and endogenous ADE2 fragment are indicated. Cleavage by HO endonuclease releases a short fragment (cut product) that is elongated by telomerase (de novo telomere healing). (C) Exceptional de novo telomere addition events. Sequences added to the 5′-TG-3′ primer within the HO endonuclease cleavage site are shown in a 5′ to 3′ orientation. The first core motif is underlined and the TG sequence generated by HO endonuclease cleavage is italicized. (D) Number of TG dinucleotide sequences synthesized between the 5′-TG-3′ HO cleavage site and the first core motif by EST2 or est2-LT^E76K telomerase. Total de novo telomeres analyzed were 17 for EST2 and 16 for est2-LT^E76K. (E) Model of the preferred alignment for EST2 and est2-LT^E76K telomerase with the primer remaining after HO endonuclease cleavage (indicated in gray italics). Newly synthesized sequence extends to the right. The first core motif is underlined.