Fig. 9
Effects of TEL-patch mutants in tpz1-AWAAA mutant. a Sequence alignment of the TEL-patch region of TPP1 from human and mouse and Tpz1 from four Schizosaccharomyces species. Identical residues conserved among four or greater species are marked black, while amino acid residues that maintain similar chemical properties are marked gray. TEL-patch residues in Tpz1 mutated in this study and potentially equivalent sites in human TPP1 are also indicated. b, c Southern blot analysis to test effect of b K75A or c T78A in AWAAA mutants with or without Tpz1-Stn1 fusion on telomere length. All genomic DNA samples were prepared from cells that have been extensively restreaked (>150 generations) on YES plates. While strains used in Southern blot analysis did not carry an epitope tag, corresponding myc-tagged versions of wild-type and mutants (with or without Stn1 fusion) were used in western blot and co-IP experiments, and results indicated that none of the mutant combinations greatly affected protein expression or disrupted interaction of Tpz1 or Tpz1-Stn1 to Poz1, Pot1 or Ccq1 (Supplementary Fig 5b and 8). d, e Dot-blot ChIP assays from asynchronous cell cultures to monitor binding of d Ten1 and e Trt1TERT at telomeres. Expression levels of myc-tagged proteins used in ChIP assays were monitored by western blot analysis. Anti-Cdc2 blots served as loading control. Molecular weight (kDa) of size markers are indicated. Plots show mean values plus/minus SEM and distribution of individual data points from at least six independent experiments. Raw data values and statistical analysis of ChIP assays by two-tailed Student’s t-test are shown in Supplementary Data 1
