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Initiation of chromosomal DNA replication which is stimulated without oversupply of DnaA protein in Escherichia coli

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Summary

The temperature-sensitive dnaA46 mutation in Escherichia coli can be phenotypically suppressed at 42° C by oversupply of GroELS proteins, and the suppressed cells grow extremely slowly at 30° C. We found that the phenotype of dnaA46 showing this cold sensitivity was dominant over the phenotype of dnaA +, and could not be rescued by introduction of oriC-independent replication systems. These results suggest that the cold sensitivity was not caused by a simple defect in replication. When a growing culture of a dnaA46 strain with a GroELS-overproducing plasmid was shifted from 42° to 30° C in the presence of chloramphenicol, the chromosomal DNA replicated excessively. Initiation of replication occurred at the site of oriC repeatedly four or five times during a 4 h incubation period without concomitant protein synthesis, indicating an excessive capacity for initiation. Such overreplication did not take place at 42° C in the suppressed dnaA46 strain, or at either temperature in GroELS-oversupplied dnaA + cells. No significant difference was detected between the cellular content of DnaA protein in suppressed cells where the initiation capacity was abnormally high, and that in wild-type cells in which the initiation capacity was normal. Thus, DnaA protein might function in vivo through some phase control mechanism for initiation, apart from a simple regulation by its total amount. A possible mechanism is proposed based on the participation of GroELS proteins in protein folding.

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Author notes

  1. Tsutomu Katayama

    Present address: Department of Biochemistry, Stanford University, 94305, Stanford, CA, USA

Authors and Affiliations

  1. Institute for Virus Research, Kyoto University, 606, Kyoto, Japan

    Tsutomu Katayama & Toshio Nagata

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  1. Tsutomu Katayama
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  2. Toshio Nagata
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Communicated by M. Sekiguchi

A preliminary account of this work was presented at the Annual Meeting of the Molecular Biology Society of Japan in 1989.

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Katayama, T., Nagata, T. Initiation of chromosomal DNA replication which is stimulated without oversupply of DnaA protein in Escherichia coli . Molec. Gen. Genet. 226, 491–502 (1991). https://doi.org/10.1007/BF00260663

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  • DOI: https://doi.org/10.1007/BF00260663

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