The mutant DnaAcos protein which overinitiates replication of the Escherichia coli chromosome is inert to negative regulation for initiation

Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30 °C. DnaAcos protein was purified from an overproducing strain and found to be as active as wild-type DnaA protein in initial synthesis rates of minichromosome replication in vitro at 30 °C. However, whereas efficient initiation occurred for only 20 min with wild-type DnaA protein, it continued for 45 min with DnaAcos protein, an indication that DnaAcos protein retained initiation activity for a longer time than wild-type DnaA protein. Also, whereas wild-type DnaA protein is inactivated by ADP binding, DnaAcos protein failed to be inactivated by ADP due to its inability to bind nucleotide. Thus, DnaAcos protein appears to lack negative regulation for its initiation activity. At 42 °C, a temperature at which initiation of chromosome replication is normal in the dnaAcos mutant, in vitro DnaAcos protein activity decreased to 25% of that observed at 30 °C. This coincident occurrence of normal initiation in vivo and reduced activity is consistent with the idea that negative control of DnaA protein activity is necessary for normal replication.