FIGURE 3.

UvrD helicase activity on a DNA substrate containing DPC in the translocating strand. A, UvrD helicase activity on the non-damaged (50T-30T) and the damaged duplex substrate containing a DPC in the translocating strand. The ³²P-labeled 30-mer oligodeoxynucleotide (30T) was annealed to the complementary 50-mer oligodeoxynucleotide (50T), and T4-pdg was covalently linked to the 50-mer strand. Reaction mixtures containing 1 nm concentrations of the indicated duplex DNA substrate and specified concentrations of UvrD were incubated at room temperature for 5 min under standard conditions. The asterisk indicates the 5′ of the ³²P-labeled strand of the duplex substrate. B, quantitation of the experiments in A. Percentage duplex substrate is graphically represented as a function of UvrD concentration used. The error bars indicate the S.D. derived from three independent helicase reactions. C, reactions with UvrD helicase at higher concentrations (0–40 nm) and a longer incubation time (15 min). Standard helicase reactions were run with the non-damaged duplex (50T-30T) and damaged substrate containing a DPC in the translocating strand. The percentage of duplex substrate is graphically represented as a function of the UvrD concentration. The error bars indicate the S.D. derived from three independent helicase reactions. The abbreviations ND, ss DNA, ds DNA (±AP site), and ds DNA-DPC correspond to the non-damaged, single-stranded DNA, double-stranded DNA with or without AP site, and double-stranded DNA containing a DPC, respectively.