The interplay of the GrpE heat shock protein and Mg2+ in RepA monomerization by DnaJ and DnaK.
Genetic and biochemical studies have established that the sole function of the Escherichia coli DnaJ, DnaK, and GrpE heat shock proteins in plasmid P1 DNA replication is to convert RepA dimers to monomers. Monomers bind avidly to oriP1 DNA and initiate DNA replication. However, with purified heat shock proteins, only DnaJ, DnaK, and ATP were required for the monomerization of RepA; GrpE was not required. We have found reaction conditions that mimic the physiological situation. GrpE function is absolutely necessary for RepA activation in vitro with DnaJ and DnaK when the free Mg2+ concentration is maintained at a level of approximately 1 microM by a metal ion buffer system. EDTA or physiological metabolites, including citrate, phosphate, pyrophosphate, and ATP, all elicit the GrpE requirement. With these metal ion-buffering systems, GrpE specifically lowers the concentration of Mg2+ required for the RepA activation reaction. The absence of Mg2+ blocks activation and high levels of Mg2+ in solution bypass the requirement for GrpE but not for the other two heat shock proteins. Our results imply that GrpE facilitates the utilization of Mg2+ for an essential step in RepA activation.
Skowyra
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Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Wickner
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D001426:Bacterial Proteins / Q000378:metabolism*; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins; D011756:Diphosphates / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D015636:Magnesium Chloride / Q000494:pharmacology*; D010957:Plasmids; D011506:Proteins; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011756:Diphosphates; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D015636:Magnesium Chloride; D004492:Edetic Acid; C063842:dnaK protein, E coli; D004265:DNA Helicases; D008274:Magnesium; C003319:sodium pyrophosphate