OVERINITIATION OF CHROMOSOME-REPLICATION IN THE ESCHERICHIA-COLI DNAACOS MUTANT DEPENDS ON ACTIVATION OF ORIC FUNCTION BY THE DAM GENE-PRODUCT

The activity of DnaA protein, the initiator of chromosome replication in Escherichia coil, is regulated by adenine nucleotide binding; the A TP-bound form, not the ADP-bound form, is active, DnaAcos is a mutant protein that is insensitive to negative regulation by ADP, Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30 degrees C, a restrictive temperature for growth, To determine the c ontrol factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn5 insertion mutagenesis, Three of the suppressors carried Tn5 in the ar oK or aroB gene, the first two cistrons in the dam operon, Complementa tion tests revealed that the dam gene is responsible for the suppressi on, Over-replication of the chromosome was inhibited in the dnaAcos ar oK::Tn5 double mutant, and initiation of chromosome replication in the dnaA(+) aroK::Tn5 mutant was partially inhibited, The aroK(or B)::Tn5 cells contained DnaA molecules at a level similar to that in the pare ntal aroBK(+) strain, Moreover, dnaAcos suppression depended on the fu nction of the seqA gene, Thus, Dam activity positively regulates initi ation of chromosome replication in vivo, SeqA function seems to be dis tinguished from the control of DnaA protein by adenine nucleotide bind ing.