A STIMULATION FACTOR FOR HYDROLYSIS OF ATP BOUND TO DNAA PROTEIN, THEINITIATOR OF CHROMOSOMAL DNA-REPLICATION IN ESCHERICHIA-COLI

Hydrolysis of ATP bound to DnaA protein by its intrinsic ATPase activi ty negatively controls chromosomal DNA replication in Escherichia coli . We developed a new in vitro assay system for ATP hydrolysis, which m akes feasible a search for factors affecting the ATPase activity of Dn aA protein. A crude cell extract enhanced the hydrolysis of ATP bound to DnaA protein, in a dose-dependent manner. Gel-filtration analyses r evealed a single entity of the stimulation factor for the ATP hydrolys is and an apparent molecular mass of 170 kDa. The stimulation activity for ATP hydrolysis coeluted with the inactivation activity for DnaA p rotein initiating an oriC DNA replication, as determined by anion-exch ange and gel-filtration column chromatographies. Activity of the stimu lation factor required DNA and ATP. These observations suggested that IdaA protein, a previously described negative factor for DnaA protein, inactivated DnaA. protein through stimulation of the hydrolysis of AT P-bound to DnaA protein. (C) 1998 Academic Press.