Extended Data Fig. 2. Validation of the C-strand synthesis reaction on telomeric DNA templates.

a, Product formation requires DNA template, CST–Polα-primase, and ribonucleotides (rNTPs). +pp: CST–Polα-primase. -pp: CST with Polα-primase removed by 300 mM NaCl elution. DNA templates are None, or 100 nM 3xTEL, 9xTEL and 15xTEL. Products labeled with α-³²P-dCTP. Nucleotide (nt) sizes are based on telomerase reaction with a 5’-end labelled primer (T-End). b, Longer DNA templates give larger ladders of C-strand products. Templates consist of the number of telomeric repeats indicated. 3xTEL and 4xTEL do not support DNA synthesis under these conditions. Products labeled with α-³²P-dCTP. T-Body is a marker lane of body-labelled telomerase products, which run about one nt slower than the T-End products; the T-End products have a 5’-phosphate which increases their electrophoretic mobility. c, Adding a 10-nt tail to the 5’ end of a 9xTEL template confirms that at least some products runoff the end of the template, and adding an antisense oligonucleotide to pair with the 10-nt tail shows that C-strand synthesis stops at double-stranded DNA. 'GC tail' is 5’-CGCCGCCGCC, followed by (TTAGGG)₉, and the 'anti' oligo is 5’-CTAAGGCGGCGGCG, designed to pair with the GC tail and the first four nt of the adjacent telomeric repeat. Products labelled with α-³²P-dATP. Reactions performed at two MgCl₂ concentrations as indicated. Reactions +dGTP allowed DNA synthesis to use the GC tail as template (brackets), while the sets of lanes without dGTP still gave some synthesis because of misincorporation. Addition of the anti oligo (lanes 5, 9, 14, 18) inhibited use of the GC tail as template; as a control (lanes 3, 7, 12, 16), the anti oligo had no effect with the 9xTEL template, which contains no complementary sequence. LC, loading control. d, POT1-TPP1N bound to telomeric DNA templates does not prevent CST–Polα-primase action but inhibits partially at high molar excess of POT1-TPP1N/template. Four DNA templates at 50 nM preincubated for 30 min at 30 °C with indicated concentrations of POT1-TPP1N prior to 1 h CST–Polα-primase reaction at 30 °C. Comparing 9xTEL-GC tail without and with the antisense oligonucleotide (anti) that binds the GC tail again confirms that C-strand synthesis stops when it encounters dsDNA. LC, loading control. e, POT1-TPP1N binding to trace amounts of radiolabelled 9xTEL and 15xTEL DNA as determined by electrophoretic mobility shift assay (EMSA). Binding for 30 min at 30 °C in C-strand synthesis buffer. K_d^app values ± SD (n = 3 independent binding curves). For gel source data, see Supplementary Fig. 1

Source Data