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.
Department of Biochemistry, Michigan State University, East Lansing 48824-1319.
T R
TR
Hupp
J M
JM
Kaguni
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H; C063842:dnaK protein, E coli; D004264:DNA Topoisomerases, Type I