The Rep proteins of some plasmid replicons have two functions. Dimers bind to the operator sequences acting as auto-repressors, whereas monomers bind to the iterons to initiate replication of DNA. The ColE2 Rep proteins are present mostly in a dimeric form with some multimers larger than dimers in solution, while the form of Rep binding to Ori is not known. We used an EMSA-based method to determine the molecular weight of Rep in the Rep-Ori complex. The result suggested that Rep binds to Ori as a monomer. In addition, the result of EMSA using the Rep protein fused with the maltose binding protein and the HiS(6)-tag also supported this conclusion. We proposed that dimerization of Rep might probably be involved in keeping the copy number of the ColE2 plasmid at the normal low level by limiting the amount of active monomeric forms of Rep in the host cell. (c) 2006 Elsevier Inc. All rights reserved.