Figure 6.

DnaB helicase activity for an unwound oriC-mimic gapped DNA. A, structure of the unwound oriC-mimic gapped DNA and a conceivable mechanism for DnaB helicase loading. Orange box, DnaA box R1; *, the ³²P-labeled site. DnaA is shown as illustrated in Fig. 1A. The sequence of the gapped DNA is the same as that of the region spanning from DnaA box R1 to the DUE, except for the deletion of the DUE 28-mer sequence in the upper strand (see the conceivable structure of the left-half oriC open complex in Fig. 1A). DnaA binds to the DnaA box on the gapped DNA and recruits the DnaB–DnaC complex via DnaA domain I, which promotes DnaB loading onto the ssDNA region and stimulates helicase function. B, gapped DNA (10 fmol, 1.1 nm) was incubated on ice for 5 min with DnaA (0–0.5 pmol or 55 nm), followed by further incubation at 37 °C for 8 min in the presence or absence of DnaB (WT or L160A) and DnaC that had been incubated on ice for 10 min. DNA was analyzed as described in the legend to Fig. 5B. Error bars, S.D.