FIGURE 2.

F231S and W233L are thermolabile in DNA replication of an oriC-containing plasmid and M13 A site ssDNA. Reactions containing 1 or 2 mm ATP, as indicated, were assembled with a supercoiled plasmid carrying oriC (M13oriC2LB5) (A and B), or a ssDNA bearing a DnaA box in a hairpin structure (M13 A site) (C–F), purified replication proteins, and the indicated amounts of DnaC, F231S, or W233L as described under “Experimental Procedures.” After incubation for 20 (A and B) or 10 min (C–F) at 30 or 37 °C, DNA synthesis (as pmol of acid-insoluble radioactivity) was measured by liquid scintillation spectrometry. Similar results to those presented were obtained in at least three essentially identical experiments.