Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin.
Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter. Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression. In this study, using in vivo dimethyl sulfate footprinting, we have confirmed the roles of the three heat shock proteins in promoting RepA binding to the origin. The defects in both activities could be suppressed by increasing the concentration of wild-type RepA over the physiological level. We also isolated RepA mutants that were effective initiators and repressors without requiring the heat shock proteins. These data suggest that the heat shock proteins facilitate both repression and initiation by promoting only the DNA-binding activity of RepA. In a similar plasmid, F, initiator mutants that confer heat shock protein independence for replication were also found, but they were defective for repression. We propose that the initiator binding involved in repression and the initiator binding involved in initiation are similar in P1 but different in F.
Sozhamannan
S
S
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
Chattoraj
D K
DK
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017101:Bacteriophage P1 / Q000235:genetics / Q000254:growth & development*; D001483:Base Sequence; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins; D017422:Sequence Analysis, DNA; D015534:Trans-Activators; D014170:Transformation, Genetic; D014779:Virus Replication / Q000187:drug effects*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; C063842:dnaK protein, E coli; D004265:DNA Helicases