I have been interested in the initiation of DNA replication and the regulation of replication and have been using an in vitro replication system that replicates DNA carrying the plasmid PI origin of replication. This reaction requires the Pl RepA initiator protein, which binds to five sites in the PI origin, and many host proteins including, DnaA, DnaB, DnaC, DnaG, RNA polymerase, DNA gyrase and DNA polymerase HI. I have found that three of the E. coli heat shock proteins, DnaJ, DnaK (the hsp7O homolog) and GrpE are also required. In collaboration with J. Hoskins and K. McKenney, we have found that DnaJ and DnaK, in an ATP dependent reaction, stimulate the Pl origin specific DNA binding activity of RepA by about 100-fold. We have shown that the mechanism of activation is the conversion of RepA dimers into monomers and that the monomer form binds with high affinity to Pl origin DNA. Reversible chemical denaturants also convert RepA dimers to monomers and simultaneously activate Pl origin DNA binding. Increasing protein concentration converts monomers to dimers and deactivates RepA. We have also been studying the regulation of Pl DNA replication. The plasmid control region contains nine repeats of the RepA binding site. However, the simple explanation of regulation by titration of RepA has been ruled out by in vivo experiments and our observation that DNA containing the origin and the control locus is not replicated in vitro even when RepA is supplied in excess relative to binding sites. RepA can bind simultaneously to the origin and the control region causing the DNA to loop in vitro, suggesting that DNA pairing may regulate DNA replication. By rearranging the orientation and the spacing of the RepA sites in the control region and by characterizing the mutant RepA proteins, we hope to understand the mechanism of regulation of replication.
