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Journal of Bacteriology, February 2007, p. 1061-1071, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.01455-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Specific Interaction between the Initiator Protein (Rep) and Origin of Plasmid ColE2-P9

Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano 390-8621, Japan

Received 14 September 2006/ Accepted 1 November 2006

The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.

Published ahead of print on 10 November 2006.

This article has been cited by other articles:

• Aoki, K., Shinohara, M., Itoh, T. (2007). Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38. J. Bacteriol. 189: 2392-2400 [Abstract] [Full Text]