Mechanism of origin activation by monomers of R6K-encoded pi protein.
One recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific Rep proteins that bind to DNA sequences called iterons. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, pi, with seven tandem iterons of gamma ori. Remarkably, both pi monomers and pi dimers can bind to iterons, a new paradigm in replication control. Dimers, the predominant form in the cell, inhibit replication, while monomers facilitate open complex formation and activate the ori. Here, we investigate a mechanism by which pi monomers out-compete pi dimers for iteron binding, and in so doing activate the ori. With an in vivo plasmid incompatibility assay, we find that pi monomers bind cooperatively to two adjacent iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two iterons but is diminished only slightly by insertion of a full helical turn between two iterons. These studies show also that pi bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. pi monomer/iteron interactions were quantified using a monomer-biased pi variant in vitro with the same collection of two-iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. pi dimer/iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that pi dimers bind with negligible cooperativity to two tandem iterons.
Bowers
Lisa M
LM
Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, USA.
Krüger
Ricardo
R
Filutowicz
Marcin
M
D001665:Binding Sites; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D018741:Replication Origin; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases