THE LOCALIZED MELTING OF MINI-F ORIGIN BY THE COMBINED ACTION OF THE MINI-F INITIATOR PROTEIN (REPE) AND HU AND DNAA OF ESCHERICHIA-COLI

Replication of mini-F plasmids requires the initiator protein RepE, wh ich binds specifically to four iterons within the origin (ori2), as we ll as some host factors that are involved in chromosomal DNA replicati on. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Es cherichia coli proteins DnaA and HU on the localized melting of ori2 D NA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50 degrees bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting wit hin the ori2 region, as detected by sensitivity: to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around t he 13mer of ori2, whose sequence closely resembles the set of 13mers f ound in the chromosomal origin oriC. Further addition of DnaA to the r eaction mixture increased the efficiency of melting and appeared to ex tend melting to the adjacent AT-rich region. Moreover, DNA melting wit h appreciably higher efficiencies was observed with mutant forms of Re pE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE bi nding and, with the assistance of HU, induces a localized melting in t he 13mer region. The addition of DnaA extends melting to the AT-rich r egion, which could then serve as the entry site for the DnaB-DnaC comp lex, much as has been documented for oriC-dependent replication.