Extended Data Fig. 9. CST–Polα-primase purified from HEK cells is much more active on telomeric DNA templates than recombinant human Polα-primase purified from insect cells.

a, Under two reaction conditions (low, 0.2 mM rATP and 1 mM MgCl₂; high, 1.0 mM rATP and 2 mM MgCl₂), CST–Polα-primase purified from HEK cells (HEK CST-PP) is more than 10,000x more reactive than human Polα-primase purified from insect cells (Insect PP). For example, compare 1000 nM Insect PP with 1 nM HEK CST-PP under the same reaction conditions: lanes 11 and 19, lanes 20 and 28, lanes 23 and 31. Phosphorimager counts are given for those lanes that had a signal above background. b, The extremely large difference in activity between CST-PP and PP also pertains to the 9xTEL and 15xTEL templates using the 'high' reaction conditions of a (1.0 mM rATP and 2 mM MgCl₂). In both a and b, template DNA was 100 nM. c, Reactions under the conditions of ref. ³⁸, with 50-fold higher DNA template concentrations (5 µM). For the 15xTEL template, the difference in activity was greater than 10-fold; compare the two 10 nM reactions, lanes 7 and 9; or 100 nM Insect PP with 10 nM HEK CST-PP, lanes 5 and 9; or 50 nM Insect PP with 5 nM HEK CST-PP, lanes 6 and 10. However, the processivity of the two enzymes was similar; compare lanes 5 and 6 with lanes 9 and 10. (The high-concentration Insect PP points in lanes 2–4 show re-priming, not high processivity.) For the dT72 template, there was very little difference in activity under these conditions; compare the two 10 nM reactions, lanes 6 and 8, or the two 5 nM reactions, lanes 7 and 9. Note that the HEK CST-PP is somewhat more processive than the Insect PP; compare dT72 lanes 8-11 with lanes 4-7. (The high-concentration Insect PP points (lanes 1-3) show re-priming, not high processivity.) For this entire figure, note that the HEK CST-PP concentrations are based on the concentration of CST, so the concentration of PP in these preparations is 5-fold lower (see Extended Data Fig. 1). Thus, in terms of PP, the large activity advantage of CST-PP indicated above is actually 5-fold larger. Each gel contains a telomerase ladder (T-End) as markers. Each lane contains the same loading control (LC), a labelled 16 nt DNA. For gel source data, see Supplementary Fig. 1.