Figure 2.

DnaB pulldown assay for oriC loading activity. IHF (0.5 pmol, 61 nm) and oriC plasmid pBSoriC (17 fmol, 2.1 nm) were incubated at 37 °C for 15 min with (+) or without (−) ATP–DnaA (1 pmol, 120 nm), DnaC (2 pmol, 240 nm), and His-DnaB (2 pmol, 240 nm, as monomer). His-DnaB–bound pBSoriC was collected with Co²⁺-conjugated magnetic beads, washed with 100 mm NaCl, and analyzed by 1% agarose gel electrophoresis. Quantitative standards (Std) of pBSoriC were included in the same gel. The percentages of recovered DNA relative to the input DNA are indicated as Recovered DNA (%) (means ± S.D. (error bars); n = 2). Representative gel images are also shown. The position of pBSoriC (pBS; supercoiled form) is indicated. His-DnaB WT and the indicated mutant derivatives, L158A (A), L160A (B), R164A (C), V165A (D), I168A (E), and V190A (F), were used for this experiment.