Initiation of DNA replication in Delta cya mutants of Escherichia coli K12

The purified DnaA protein has a high affinity for cyclic AMP (cAMP). Using equilibrium dialysis, we determined the K-A value for cAMP as 0.819 mu M-1. The number of cAMP binding sites per DnaA protein molecule was calculated to be 1.04. This binding was quite specific for cAMP. ATP was also bound by DnaA protein and inhibited cAMP binding. This inhibition was non-competiti ve in nature with an inhibition constant (K-i) of about 8.25 mu M. However, in vivo we have found not only that the DnaA protein level is reduced in a cyclase deletion mutant strain, Delta cya, but also that DnaA protein is n ot degraded. The Delta cya mutants of E. coil are unable to continue DNA sy nthesis in the absence of de novo protein synthesis and the initiation of D NA replication in these mutants takes place from oriC. (C) 1999 Societe fra ncaise de biochimie et biologie moleculaire/Editions scientifiques et medic ales Elsevier SAS.