C OMMUNICATION Cooperative Binding Mode of the Inhibitors of R6K Replication, π Dimers Lisa M. Bowers and Marcin Filutowicz⁎ Department of Bacteriology, University of Wisconsin- Madison, 1550 Linden Drive, Madison, WI 53706, USA Received 24 September 2007; received in revised form 24 December 2007; accepted 15 January 2008 Available online 26 January 2008 The replication initiator protein, π, plays an essential role in the initiation of plasmid R6K replication. Both monomers and dimers of π bind to iterons in the γ origin of plasmid R6K, yet monomers facilitate open complex forma- tion, while dimers, the predominant form in the cell, do not. Consequently, π monomers activate replication, while π dimers inhibit replication. Re- cently, it was shown that the monomeric form of π binds multiple tandem iterons in a strongly cooperative fashion, which might explain how mo- nomers outcompete dimers for replication initiation when plasmid copy number and π supply are low. Here, we examine cooperative binding of π dimers and explore the role that these interactions may have in the inac- tivation of γ origin. To examine π dimer/iteron interactions in the absence of competing π monomer/iteron interactions using wild-type π, constructs were made with key base changes to each iteron that eliminate π monomer binding yet have no impact on π dimer binding. Our results indicate that, in the absence of π monomers, π dimers bind with greater coopera- tivity to alternate iterons than to adjacent iterons, thus preferentially leaving intervening iterons unbound and the origin unsaturated. We discuss new insights into plasmid replication control by π dimers. © 2008 Elsevier Ltd. All rights reserved. Edited by J. Karn Keywords: cooperativity; plasmid replication; R6K; Rep-iteron interaction; replication control It is believed that all naturally occurring plasmids employ efficient copy-control mechanisms to ensure their maintenance at a reasonably constant copy number from cell to cell. The antibiotic resistance plasmid, R6K, is maintained at a steady 15–20 copies per chromosome 1 in a wide variety of bacterial hosts. 2 For this to occur, regulatory controls at the step of replication initiation work to increase low plasmid copy numbers and to reduce elevated ones. 3 Controlled replication of plasmid R6K requires two plasmid-encoded elements: the iterons in the γ origin of replication (ori) and the pir gene that en- codes the replication (Rep) protein, π (Fig. 1) 5–9 γ ori activation requires the binding of monomers of π protein to the seven 22 base-pair (bp) iterons within γ ori that are adjacent to an A+T-rich region of the plasmid. 10–12 The binding of π monomers to iterons causes an apparent bending of the origin DNA, allowing the nearby A+T-rich region to melt, the replication complex to bind, and replication to start unidirectionally from a specific site within the A + T- rich region. 12–14 While monomers of π activate replication, dimers of π appear to inhibit replication through several different mechanisms (Fig. 1). 10,15–18 π dimers bind a non-iteron site within the A+T-rich region in proxi- mity to the start sites for leading strand synthesis. 19 It has been hypothesized that π dimers negatively modulate the priming step of the replication process by binding to this site. 19–21 π dimers also inactivate γ ori by binding iterons. π was the first of its family of Rep proteins that was shown to be capable of binding iterons as a dimer. 10 Since then, two more Reps were also shown or inferred to bind iterons as dimers, thus establishing a new trend in Rep/iteron control. 22–24 Although a π dimer can bend iteron DNA to the same extent as a π monomer, 13 π dimers *Corresponding author. E-mail address: msfiluto@wisc.edu. Abbreviations used: γ ori, γ origin of replication; bp, base pair; WH2, C-terminal winged helix; WH1, N-terminal winged helix; wt, wild type; IHF, integration host factor. Available online at www.sciencedirect.com doi:10.1016/j.jmb.2008.01.039 J. Mol. Biol. (2008) 377, 609–615 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.