Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the ␥ origin of plasmid R6K, the Rep protein, , is distinctive in that it can bind the seven 22-bp iterons in two forms; monomers activate replication, whereas dimers act as inhibitors. In this work, we used wild type and variants of the protein with altered monomer/dimer ratios to study iteron/ interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by monomers and dimers. We also isolated iteron mutants that affected the binding of monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, monomers interact with nucleotides spanning the entire length of the iteron. In contrast, dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.

The focus of this minireview is the replication control of the 39.9-kb plasmid R6K and its derivatives. Historically, this plasmid was thought to have a narrow host range but more recent findings indicate that its derivatives can replicate in a variety of enteric and non-enteric bacterial species (Wild et al., 2004). In the four-plus decades since it was first described, R6K has proven to be an excellent model for studies of plasmid DNA replication. In part this is because of its similarities to other systems in which replication is activated and regulated by Rep protein and iteron-containing DNA. However its apparent idiosynchracies have also added to its significance (e.g., independent and co-dependent replication origins, and Rep dimers that stably bind iterons). Here, we survey the current state of knowledge regarding R6K replication and place individual regulatory elements into a proposed homeostatic model with implications for the biological significance of R6K and its multiple origins of replication.

One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is "handcuffing," in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded protein with the seven 22-bp iterons of the ␥ origin of replication. Like other related Rep proteins, exists as both monomers and dimers. However, the ability of dimers to bind iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind iterons. Here, we describe experiments to determine if monomers or dimers of protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (⅐P106L ∧ F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of protein (⅐M36A ∧ M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type . The data suggest that dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the ␥ ori system. Thus, dimers of appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of expression, in cis competition (for origin binding) with monomeric activator , and handcuffing-mediated inhibition of replication in trans.

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