Fig. S3.

Formation of the pol δ holoenzyme is independent of preincubation time. (A) Schematic representation of experiment performed to monitor extent of holoenzyme formation with increasing preincubation time. (B) 16% denaturing sequencing gel of the primer extension products for pol δ alone (lanes 1–7) and a pol δ holoenzyme assembled with PCNA (lanes 8–14) in the presence of 250 µM of each dNTP and 0.5 mM ATP. The size of the substrate and full-length product are indicated on the left. As a control for each condition, an aliquot was removed after 70 s of preincubation and quenched before the addition of dNTPs and trap (lanes 7 and 14). (C) Quantification of the primer-extension products (total, ■) and the fully-extended primer (●) for holoenzymes assembled with PCNA. Data points were fit to a flat line. As can be seen, the ratio of full-length product to the total amount of primer extension products was maintained when the preincubation time was increased. This demonstrates that the two populations of pol δ holoenzymes are independent of the preincubation time and are not interconvertible. (D) Quantification of the probability of insertion for steps 2 through 6 for holoenzymes assembled with PCNA. Data points were fit to a flat line.