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Nucleic Acids Research, 1991, Vol. 19, No. 11 2875-2880
© 1991

MOLECULAR BIOLOGY

Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication

Mitsuru Kido+, Hisashi Yasueda§ and Tateo Itoh*

Laboratory of Genetics, Department of Biology, Faculty of Science, Osaka University Toyonaka, Osaka 560, Japan

*To whom correspondence should be addressed

Received March 19, 1991. Revised May 13, 1991. Accepted May 13, 1991.

The product of the rep gene of ColE2 is required for initiation of ColE2 DNA replication. The rep gene was placed under the control of the promoters, PL and PR, and the heat-labile c1857 repressor of bacteriophage {lambda}. The Rep protein was identified as a 35 Kd protein by the maxicell method in combination with heat-induced expression. The protein was efficiently expressed from these promoters in unirradiated cells and accumulated up to a few per cent of the total cellular proteins. It was partially purified (about 80% pure) and its properties examined. The amino acid sequence of the amino terminal portion of the partially purified protein agreed well with that predicted from the nucleotide sequence of the rep gene. One of the characteristic features of the rep gene is frequent usage of rare codons, especially those for arginine. The protein specifically stimulated replication of ColE2 DNA but not that of ColE3 DNA in crude cell extracts of Escherichia coli. Specific binding of the protein to plasmid DNA containing the origin region of ColE2 was demonstrated by the filter binding method. Neither endonuclease activity nor topoisomerase activity was detected by using ColE2 DNA.

+Present addresses: Institute for Molecular Biology, Osaka University, Suita, Osaka 550

§Central Research Laboratories, Ajinomoto Co. Inc., Kawasaki, Kanagawa 210, Japan


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