The mechanisms by which every daughter cell in a growing bacterial culture is assured of an equivalent genetic dowry continue to be the focus of our inquiries. To this end we have been studying an inessential element of the bacterial trousseau, a stable, low-copy number plasmid, prophage P1. During the period of this report studies of plasmid replication have provided considerable molecular detail about how the P1-encoded initiator protein, RepA, and the host-encoded initiator protein, DnaA, perform their functions and where and how the host heat-shock proteins DnaJ, DnaK, and GrpE enter into the picture. Evidence has been obtained that, although they are capable of monomerizing dimeric RepA protein, the function of DnaJ, DnaK and GrpE in vivo is to perform a more subtle alteration. These proteins activate the already monomeric form of RepA for binding to specific sites. Activation for binding appears to be the unique role of the heat shock proteins in P1 replication. Binding of the activated RepA monomers to sites in the P1 origin along one face of the DNA helix causes a right- handed wrapping of the DNA about the protein core and, through a cooperative interaction, enhances the specific binding of initiators to the origin. It is the bound DnaA protein rather than the bound RepA protein that appears to accomplish a strand opening that is presumed to be essential for primer synthesis. Studies of plasmid partitioning during the period of this report have challenged established ideas about the structure and function of the P1 partition module. The sequence of P1 parB (one of two P1 proteins required for active partition) has been corrected, a cryptic incompatibility element has been unmasked, and the interference with stabilization that occurs when two plasmids possess the same partition module (partition incompatibility) has been shown to lead to a greater loss of stability than the commonly held model of such incompatibility admits. In addition, new tools have been developed with which to discern the presently elusive features of DNA context that we find to be critical in determining whether the centromere- analog, parS, is functional or dysfunctional.
