Figure 1.

Casein trap experiment showing that one cycle of ClpA binding and ATP-dependent release activates RepA. ClpA-RepA complexes were formed by incubating 4.3 pmol of ClpA and 0.3 pmol of RepA in 20 μl of buffer A containing 1 mM ATP[γ-S] for 20 min at 23°C. Then 42.4 pmol of casein was added, followed by 1 mM ATP where indicated. After an additional 15 min at 23°C, activated RepA was measured by adding 1 μg of calf thymus DNA and 20 fmol of ³H-labeled oriP1 plasmid DNA. After 5 min at 0°C, the mixtures were filtered through nitrocellulose filters, and the retained radioactivity was measured. In control experiments ATP[γ-S] was omitted from the first reaction, casein was added to the first reaction with or without ATP[γ-S], or ATP was omitted from the second reaction, as indicated.