FIGURE 10.

Pulldown assay for complex formation of ADP-Hda, ATP-DnaA, and the DNA-loaded clamp. Apo-nHis-Hda (apo) and ADP-nHis-Hda (ADP) were conjugated to TALON beads as described for Fig. 8C. A, bead-conjugated apo-nHis-Hda or ADP-nHis-Hda (5 pmol) was incubated on ice for 5 min in buffer containing the DNA-loaded or DNA-free clamp (2 pmol as clamp), and ATP-DnaA (4 pmol) was included and incubated for 10 min. The bead-bound materials were analyzed as described for Fig. 8C. B, indicated amounts of DnaA were similarly analyzed using the pulldown assay in the presence of the DNA-loaded clamp (2 pmol as clamp) and nHis-Hda (5 pmol). C, indicated amounts of nHis-Hda were similarly analyzed using the pulldown assay in the presence of the DNA-loaded clamp (1 pmol as clamp) and ATP-DnaA (2 pmol).