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. 1976 Mar;73(3):752–756. doi: 10.1073/pnas.73.3.752

Isolation of an intermediate which precedes dnaG RNA polymerase participation in enzymatic replication of bacteriophage phi X174 DNA.

J H Weiner, R McMacken, A Kornberg
PMCID: PMC335996  PMID: 768984

Abstract

Conversion of phi X174 single-stranded DNA to the duplex replicative form (RF) in vitro requires at least 10 purified proteins. Three stages - strand initiation, elongation, and termination - comprise this conversion. We now identify a separate stage in strand initiation which precedes dnaG RNA polymerase participation. Incubation of five proteins - protein i, protein n, DNA unwinding protein, dnaB protein, and dnaC protein - with ATP and phi X174 DNA forms an intermediate which enables subsequent stages measured by DNA synthesis to proceed 20 times faster. The intermediate can be isolated in quantitative yield by gel filtration or by ultracentrifugation. Protein i and protein n are required in less than stoichiometric amounts and appear to be absent from the isolated intermediate. Whereas formation of the intermediate is sensitive to antibody to protein i and to N-ethylmaleimide (an inhibitor of protein n and dnaC protein), the intermediate itself is resistant to these reagents. DNA unwinding protein complexes the DNA in a ratio of 60 molecules per circle. Synthesis of the intermediate appears to require stoichiometric quantities of dnaB protein and dnaC PROTEin but their presence in the intermediate has not been established as yet.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bouché J. P., Zechel K., Kornberg A. dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. J Biol Chem. 1975 Aug 10;250(15):5995–6001. [PubMed] [Google Scholar]
  2. Geider K., Kornberg A. Conversion of the M13 viral single strand to the double-stranded replicative forms by purified proteins. J Biol Chem. 1974 Jul 10;249(13):3999–4005. [PubMed] [Google Scholar]
  3. Godson G. N. Evolution of phi-chi 174. Isolation of four new phi-chi-like phages and comparison with phi-chi 174. Virology. 1974 Mar;58(1):272–289. doi: 10.1016/0042-6822(74)90161-5. [DOI] [PubMed] [Google Scholar]
  4. Schaller H., Uhlmann A., Geider K. A DNA fragment from the origin of single-strand to double-strand DNA replication of bacteriophage fd. Proc Natl Acad Sci U S A. 1976 Jan;73(1):49–53. doi: 10.1073/pnas.73.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Schekman R., Weiner A., Kornberg A. Multienzyme systems of DNA replication. Science. 1974 Dec 13;186(4168):987–993. doi: 10.1126/science.186.4168.987. [DOI] [PubMed] [Google Scholar]
  6. Schekman R., Weiner J. H., Weiner A., Kornberg A. Ten proteins required for conversion of phiX174 single-stranded DNA to duplex form in vitro. Resolution and reconstitution. J Biol Chem. 1975 Aug 10;250(15):5859–5865. [PubMed] [Google Scholar]
  7. Syvanen J. M., Yang Y. R., Kirschiner M. W. Preparation of 125 I-Catalytic subunit of asparatate transcarbamylase and its use in studies of the regulatory subunit. J Biol Chem. 1973 Jun 10;248(11):3762–3768. [PubMed] [Google Scholar]
  8. Weiner J. H., Bertsch L. L., Kornberg A. The deoxyribonucleic acid unwinding protein of Escherichia coli. Properties and functions in replication. J Biol Chem. 1975 Mar 25;250(6):1972–1980. [PubMed] [Google Scholar]
  9. Westergaard O., Brutlag D., Kornberg A. Initiation of deoxyribonucleic acid synthesis. IV. Incorporation of the ribonucleic acid primer into the phage replicative form. J Biol Chem. 1973 Feb 25;248(4):1361–1364. [PubMed] [Google Scholar]
  10. Wickner S., Hurwitz J. Interaction of Escherichia coli dnaB and dnaC(D) gene products in vitro. Proc Natl Acad Sci U S A. 1975 Mar;72(3):921–925. doi: 10.1073/pnas.72.3.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Wickner W. Asymmetric orientation of a phage coat protein in cytoplasmic membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4749–4753. doi: 10.1073/pnas.72.12.4749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Wickner W., Kornberg A. A holoenzyme form of deoxyribonucleic acid polymerase III. Isolation and properties. J Biol Chem. 1974 Oct 10;249(19):6244–6249. [PubMed] [Google Scholar]
  13. Zechel K., Bouché J. P., Kornberg A. Replication of phage G4. A novel and simple system for the initiation of deoxyribonucleic acid synthesis. J Biol Chem. 1975 Jun 25;250(12):4684–4689. [PubMed] [Google Scholar]

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