Extended Data Fig. 6. RNA primers synthesized by CST–Polα-primase on three different DNA templates, directly visualized by omitting deoxynucleotides from the reaction.

Left half, production of short RNA oligonucleotides initiating with pppA on the 4xTEL and 9xTEL DNA templates. Their mobilities are consistent with 2, 3 and 4 nt, as indicated, although these sizes have not been independently confirmed. These products may represent abortive initiation by primase²⁷. Right half, the bottom of the gel containing the unincorporated ³²P-ATP was cut off to allow a longer exposure of the larger products. The 9xTEL template allowed synthesis of RNA primers of length 8 and 9 nt; size estimates have ± 1 nt uncertainty because they are based on the telomerase size markers, which have a different 5’ end and different base composition. There are also some longer products formed (14 – 27 nt), presumably indicating some ability of Polα to incorporate ribonucleotides when there are no dNTPs in the reaction. The 4xTEL template, which supported synthesis of the putative abortive initiation products, was too short to allow synthesis of the 8–9 nt primers. For gel source data, see Supplementary Fig. 1.