Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication ofmini-F plasmid in Escherichia coli

In bacteria, plasmids and some DNA viruses, DNA replication is initiated an d regulated by binding of initiator proteins to repetitive sequences. To un derstand the control mechanism we used the plasmid mini-F, whose copy numbe r is stringently maintained in Escherichia coil, mainly by its initiator pr otein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number o f mini-F plasmid in cis, Many incompatibility defective mutants carrying mu tations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F pl asmids lost the function to control the copy number, The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons doe s not rely on their capacity to titrate RepE protein. Using a ligation assa y, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of i ncC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping m ini-F plasmid at a low copy number.