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Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome

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Summary

A new round of chromosomal replication of a temperature-sensitive initiation mutant (dnaC) of Escherichia coli was initiated synchronously by a temperature shift from a nonpermissive to a permissive condition in the presence of arabinosyl cytosine. Increased amounts of nascent DNA fragments with homology for the chromosomal segment containing the replication origin (oriC) were found. The nascent DNA fragments were purified and treated with alkali to hydrolyze putative primer RNA and to expose 5′-hydroxyl DNA ends at the RNA-DNA junctions: The ends were then labeled selectively with T4 polynucleotide kinase and [γ-32P]ATP at 0°C and the terminally-labeled initiation fragments were purified by hybridization with origin probe DNAs containing one each of the constituent strands of oriC-DNA segment. The 32P-labeled initiation sites were then located at the resolution of single nucleotides in the nucleotide sequence of the oriC segment after cleavage with restriction enzymes. Two initiation sites of DNA synthesis, 37 nucleotides apart, were detected in one of the component strands of the oriC; in other words, in the strand whose 5′ to 3′ polynucleotide polarity lies counterclockwise on the E. coli genetic map. The results support the involvement of the primer RNA in the initiation of DNA synthesis at the origin of the E. coli genome and suggest that the first initiation event is asymmetric.

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Author notes

  1. Susumu Hirose

    Present address: National Institute for Basic Biology, Okazaki, 444, Japan

Authors and Affiliations

  1. Institute of Molecular Biology, Faculty of Science, Nagoya University, 464, Nagoya, Japan

    Susumu Hirose & Tuneko Okazaki

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

    Sota Hiraga

Authors
  1. Susumu Hirose
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  2. Sota Hiraga
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  3. Tuneko Okazaki
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Additional information

Communicated by G. O'Donovan

A portion of this work was published by T. Okazaki et al. in ICN-UCLA Symposia on Molecular and Cellular Biology (ed. B. Alberts) Vol. 19 (1980) pp. 429–447. Academic Press, New York

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Hirose, S., Hiraga, S. & Okazaki, T. Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome. Molec. Gen. Genet. 189, 422–431 (1983). https://doi.org/10.1007/BF00325904

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  • DOI: https://doi.org/10.1007/BF00325904

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