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

Molecular & General Genetics : MGG
Y KawasakiC Wada

Abstract

Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. 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 Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA 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 within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that ...Continue Reading

Citations

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