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Journal of Bacteriology, March 2007, p. 2392-2400, Vol. 189, No. 6
0021-9193/07/$08.00+0     doi:10.1128/JB.01695-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38{triangledown}

Kazuteru Aoki,1 Miki Shinohara,2,{dagger} and Tateo Itoh1,2*

Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano 390-8621,1 Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan2

Received 2 November 2006/ Accepted 5 January 2007

The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, {alpha} and ß, in the origins as specificity determinants and showed that when each component of the region A-site {alpha} pair and the region B-site ß pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site {alpha}. By using an electrophoretic mobility shift assay, we demonstrated that region B and site ß play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site {alpha} were involved in the efficient unwinding of several nucleotide residues around site {alpha}, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.

* Corresponding author. Mailing address: Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano 390-8621, Japan. Phone: 81-263-37-2489. Fax: 81-263-37-2560. E-mail: tateito{at}gipac.shinshu-u.ac.jp.

{triangledown} Published ahead of print on 19 January 2007.

{dagger} Present address: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

Journal of Bacteriology, March 2007, p. 2392-2400, Vol. 189, No. 6
0021-9193/07/$08.00+0     doi:10.1128/JB.01695-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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