(A) A sequence alignment of OB fold-domains between fission yeast Tpz1, human TPP1 and Oxytricha nova TEBP-β, in combination with a secondary structure prediction. Twelve conserved fission yeast Tpz1 residues subjected to mutagenesis are highlighted in blue with their identities and sequence numbers indicated above the alignment. Residues colored in red are previously identified TEL-patch residues in human TPP1 and mutations of them lead to compromised TPP1-TERT interaction. Black and grey shading indicates sequence identity and similarity, respectively. If the sequence is identical among at least 50% of species, the residues will be shaded in black. The same rule applies to sequence similarity, which is shaded in grey. (B) Southern blot analysis to measure telomere lengths using EcoRI-digested genomic DNA visualized by the telomere DNA probe for the indicated tpz1 mutant strains from successive re-streaks on agar plates. tpz1-K75E, tpz1-R76E, tpz1-I77R, and tpz1-R81E strains caused telomere shortening and tpz1-R81E cells showed the classic Ever Shorter Telomere (EST) phenotype. For the telomere length analysis southern blots presented in all the figures, the 1 kb plus marker from Invitrogen is used. Wild type cells are denoted as 'WT' in the blot. pol1+ indicates the EcoRI-digested pol1+ DNA fragment used as a loading control. (C) Double-mutant strain tpz1-R81E/trt1Δ shows EST telomere phenotype, similar to the trt1Δ single-mutant strain. (D) Double-mutant strain tpz1-R81E/poz1Δ shows progressive telomere shortening phenotype.