Activation of DnaA5 protein by GrpE and DnaK heat shock proteins in initiation of DNA replication in Escherichia coli.

DnaA5 protein, inactive in replication systems dependent on purified enzymes, was activated by addition of a crude enzyme fraction. Based on this assay, two proteins in the crude enzyme fraction were identified that confer replication activity upon DnaA5 protein in a purified enzyme system. That one is DnaK protein was suggested initially from studies in which DnaK protein stimulated another mutant form of DnaA protein in replication assays dependent on a crude enzyme fraction (Hwang, D. S., and Kaguni, J. M. (1991) J. Biol. Chem. 266, 7537-7555). The identification of DnaK protein as a required protein for activation of DnaA5 protein, and its inclusion in a reconstituted enzyme system of purified replication proteins, allowed for the partial purification of the second activating protein. GrpE protein was deduced to be the second activating protein based on three criteria. First, activity in partially purified fractions correlated with a protein similar in size to GrpE protein on a sodium dodecyl sulfate-polyacrylamide gel. Second, activity in partially purified fractions correlated with a protein that reacted with anti-GrpE antibody. Third, purified GrpE protein functionally replaced the second protein in activation of DnaA5 protein.