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 rep lication is essential for initiation of DNA replication and for autore gulatory 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 p lay a role in the RepA-DNA binding required for initiation and repress ion. In this study, using in vivo dimethyl sulfate footprinting, we ha ve confirmed the roles of the three heat shock proteins in promoting R epA binding to the origin. The defects in both activities could be sup pressed by increasing the concentration of wild-type RepA over the phy siological level. We also isolated RepA mutants that were effective in itiators and repressors without requiring the beat shock proteins. The se data suggest that the heat shock proteins facilitate both repressio n and initiation by promoting only the DNA-binding activity of RepA. I n a similar plasmid, F, initiator mutants that confer heat shock prote in independence for replication were also found, but they were defecti ve for repression. We propose that the initiator binding involved in r epression and the initiator binding involved in initiation are similar in P1 but different in F.