REPLICATION INITIATOR PROTEIN REPE OF MINI-F PLASMID - FUNCTIONAL-DIFFERENTIATION BETWEEN MONOMERS (INITIATOR) AND DIMERS (AUTOGENOUS REPRESSOR)

Replication of mini-F plasmid requires the plasmid-encoded RepE initia tor protein and several host factors including DnaJ, DnaK, and GrpE, h eat shock proteins of Escherichia coli. The RepE protein plays a cruci al role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a mark edly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric for m, unlike the wild-type RepE that is recovered in dimeric form. Gel-re tardation assays revealed that RepE54 monomers bind to ori2 (direct re peats) with a very high efficiency but hardly bind to the repE operato r (inverted repeat), in accordance with the properties of RepE54 in vi vo. Furthermore, the treatment of wild type RepE dimers with protein d enaturants enhanced their binding to ori2 but reduced binding to the o perator: RepE dimers were partially converted to monomers, and the ori 2 binding activity was uniquely associated with monomers. These result s strongly suggest that RepE monomers represent an active form by bind ing to ori2 to initiate replication, whereas dimers act as an autogeno us repressor by binding to the operator. We propose that RepE is struc turally and functionally differentiated and that monomerization of Rep E dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replic ation.