Three Escherichia coli heat shock proteins are required for P1 plasmid DNA replication: formation of an active complex between E. coli DnaJ protein and the P1 initiator protein.
DNA containing the plasmid origin of bacteriophage P1 is replicated in vitro by a protein fraction prepared from uninfected Escherichia coli supplemented with purified P1 RepA protein. It has previously been shown that the reaction required the E. coli DnaA initiator protein, the DnaB helicase, DnaC protein, RNA polymerase, and DNA gyrase. I show here that three E. coli heat shock proteins, DnaJ, DnaK, and GrpE, are directly involved in P1 plasmid replication. Purified DnaJ, DnaK, and GrpE proteins were required to stimulate P1 plasmid ori DNA-dependent replication in in vitro complementation assays in which the host protein fractions were prepared from cells mutated in the corresponding gene. I have also found that the DnaJ and RepA proteins form a complex. This complex exists in crude cell extracts and can be isolated as a molecular species of about 160,000 Da containing one dimer of DnaJ protein and one dimer of RepA. The complex can also be reconstituted by mixing purified DnaJ and RepA proteins. These results imply that the DnaJ-RepA complex, DnaK, and GrpE are directly involved in P1 plasmid replication.
Wickner
S H
SH
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003090:Coliphages / Q000235:genetics* / Q000378:metabolism; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D010957:Plasmids; D011506:Proteins; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases