The remarkable stability of the low copy number plasmid P1 results from a combination of mechanisms under continuing study: replication control, active partition, and plasmid addiction. Previous work had shown that plasmid replicons with shared requirements for particular bacterial proteins (DnaA, Dam) can exhibit surprising differences in the manner in which these proteins are used. Work performed in the period of the present report supports the extension of this caveat to include the three proteins DnaJ, DnaK, GrpE. These heat shock proteins appear to play a simpler role in Pl replication and one more accessible to analysis than that previously established for their role in the replication of phage lambda. The separate functions of the PI protein, RepA, in initiation of DNA synthesis and in mediation of copy number control by incA are being dissected with in vitro techniques. Two-dimensional gel electrophoresis assays provide direct information about DNA conformation. Sequence determinations of RepA binding sites generated by """"""""dirty bottle"""""""" synthesis and subjected to informational analysis provide indications that conserved regions of DNA sequence in binding sites may serve different functions. In addition to mechanistic studies of protein-DNA interactions, studies of a """"""""back-to-basics"""""""" kind are being undertaken. These test the range of validity of the functional independence of active partition and replication. In particular, newly recognized context effects that can render the PI partition system destabilizing are being explored. The nature of an addiction system in PI which appears unlike those found associated with other plasmids is also being studied.
