Figure 2.

Supplementing conditions after initial unwinding of DNA containing a reversible interstrand cross-link. (A) A second replication fork model (OD3:5′-[³²P]-OD4, 10 nM) containing a cross-link formed by bisQMP and a radiolabel on the excluded strand was incubated as described in Figure 1 for 30 min before supplementing with another aliquot of either T7gp4 (55 nM monomer) or dTTP (1 mM). Incubations were continued at 37 °C for the indicated times and then quenched with EDTA (40 mM). (B) Reaction products were detected after separation by denaturing PAGE (10%) and quantified relative to total radiolabel (%). Data represent three replicates and average values are indicated by the cross-bars. The control entitled spontaneous hydrolysis contained no T7gp4 or dTTP throughout the incubations and that entitled no supplement contained the standard concentrations of T7gp4 and dTTP in the initial incubation but no additional T7gp4 or dTTP in the subsequent incubation.