Figure 5.

Processivity of Pol η on nondamaged and damaged substrates. (A) Processive DNA synthesis by Pol η on an undamaged DNA template (substrate S-1; Fig. 1) resulting from a single binding event. In lanes 1–4, Pol η (20 nM) was preincubated with the undamaged DNA substrate (10 nM) for 15 min at 25°C, and reactions were initiated by the addition of all four dNTPs (200 μM each), MgCl₂ (5 mM), and the sonicated herring sperm DNA trap (1 mg/ml). Reactions were stopped after the indicated times, and the samples were examined by denaturing PAGE. The unextended primer (n = 0) and the extended primers (n = 1–10) are indicated. In lanes 5–8, Pol η was preincubated with the DNA substrate and with the DNA trap, and the reaction was initiated by the addition of dNTPs and MgCl₂. (B) Processive DNA synthesis by Pol η on the damaged DNA template (substrate S-3; Fig. 1) resulting from a single DNA-binding event. Reactions were performed as described for the undamaged DNA template. (C) Percentage of active Pol η molecules at each position along the template. The black bars represent the active polymerases on the nondamaged DNA template and the gray bars represent the active polymerases on the damaged template.