Identification of a Plasmid-coded Protein Required for Initiation of ColE2 DNA Replication

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1991 Jun 11

PMID 1829159

Citations 15

Authors

M Kido

H Yasueda

T Itoh

Affiliations

Soon will be listed here.

Abstract

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 cl857 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.

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Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.

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Specific interaction between the initiator protein (Rep) and origin of plasmid ColE2-P9.

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