Activation of mutant forms of DnaA protein of Escherichia coli by DnaK and GrpE proteins occurs prior to DNA replication.
Mutant forms of DnaA protein, inert in a replication system composed of other purified proteins, are "activated" by DnaK and GrpE heat shock proteins (Hupp, T. R., and Kaguni, J. M. (1993) J. Biol. Chem. 268, 13137-13142). The effect of these heat shock proteins on DnaA5 and DnaA46 protein was separated from the event of DNA synthesis by incubation in two stages. Components necessary during the first stage for "activation" included GrpE and DnaK proteins, ATP at 0.2 mM or greater, and polyvinyl alcohol (8%) or glycerol, optimal at concentrations between 20 and 30%. An ATP regenerating system provided by creatine kinase and creatine phosphate was stimulatory. Addition of the activated form of DnaA5 or DnaA46 protein to a reconstituted system containing other purified replication proteins during the second stage of incubation resulted in DNA replication. Activation of DnaA5 or DnaA46 protein by heat shock proteins was thermolabile, suggesting that the temperature sensitivity of dnaA5 and dnaA46 mutants is related to this thermolabile interaction. A third heat shock protein, DnaJ protein, interfered with the activation of DnaA5 protein if present during the first stage of incubation. This inhibitory effect was less striking if included during the second stage of incubation. These results suggest a mechanism for regulation of the activity of DnaA protein.
Department of Biochemistry, Michigan State University, East Lansing 48824-1319.
T R
TR
Hupp
J M
JM
Kaguni
D000255:Adenosine Triphosphate / Q000378:metabolism; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D009154:Mutation
D000911:Antibodies, Monoclonal; 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; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli