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Journal of Bacteriology, June 2005, p. 3779-3785, Vol. 187, No. 11
0021-9193/05/$08.00+0     doi:10.1128/JB.187.11.3779-3785.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of Dimers in Coupling ("Handcuffing") of Plasmid R6K's ori Iterons

Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, Wisconsin 53706,¹ Institute for Molecular Virology, University of Wisconsin-Madison, 1525 Linden Drive, Madison, Wisconsin 53706²

Received 16 December 2004/ Accepted 14 February 2005

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 (·P106LF107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of protein (·M36AM38A), 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.