Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1977 Jan;74(1):193–197. doi: 10.1073/pnas.74.1.193

A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication.

J F Scott, S Eisenberg, L L Bertsch, A Kornberg
PMCID: PMC393224  PMID: 138139

Abstract

The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.

Full text

PDF
193

Images in this article

197Image
on p.197

Selected References

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

  1. Abdel-Monem M., Dürwald H., Hoffmann-Berling H. Enzymic unwinding of DNA. 2. Chain separation by an ATP-dependent DNA unwinding enzyme. Eur J Biochem. 1976 Jun 1;65(2):441–449. doi: 10.1111/j.1432-1033.1976.tb10359.x. [DOI] [PubMed] [Google Scholar]
  2. Baas P. D., Jansz H. S. Bacteriophage phiX174 DNA synthesis in a replication-deficient host: determination of the origin of phiX DNA replication. J Mol Biol. 1976 Apr 15;102(3):633–656. doi: 10.1016/0022-2836(76)90339-9. [DOI] [PubMed] [Google Scholar]
  3. Cairns J., Denhardt D. T. Effect of cyanide and carbon monoxide on the replication of bacterial DNA in vivo. J Mol Biol. 1968 Sep 28;36(3):335–342. doi: 10.1016/0022-2836(68)90159-9. [DOI] [PubMed] [Google Scholar]
  4. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  5. Eisenberg S., Denhardt D. T. Structure of nascent phiX174 replicative form: evidence for discontinuous DNA replication. Proc Natl Acad Sci U S A. 1974 Mar;71(3):984–988. doi: 10.1073/pnas.71.3.984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eisenberg S., Scott J. F., Kornberg A. An enzyme system for replication of duplex circular DNA: the replicative form of phage phi X174. Proc Natl Acad Sci U S A. 1976 May;73(5):1594–1597. doi: 10.1073/pnas.73.5.1594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eisenberg S., Scott J. F., Kornberg A. Enzymatic replication of viral and complementary strands of duplex DNA of phage phiX174 proceeds by seprate mechanisms. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3151–3155. doi: 10.1073/pnas.73.9.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Francke B., Ray D. S. Formation of the parental replicative form DNA of bacteriophage phi-X174 and initial events in its replication. J Mol Biol. 1971 Nov 14;61(3):565–586. doi: 10.1016/0022-2836(71)90065-9. [DOI] [PubMed] [Google Scholar]
  9. Henry T. J., Knippers R. Isolation and function of the gene A initiator of bacteriophage phi-chi 174, a highly specific DNA endonuclease. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1549–1553. doi: 10.1073/pnas.71.4.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hill T. L. A proposed common allosteric mechanism for active transport, muscle contraction, and ribosomal translocation. Proc Natl Acad Sci U S A. 1969 Sep;64(1):267–274. doi: 10.1073/pnas.64.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ikeda J. E., Yudelevich A., Hurwitz J. Isolation and characterization of the protein coded by gene A of bacteriophage phiX174 DNA. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2669–2673. doi: 10.1073/pnas.73.8.2669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Inoue-Yokosawa N., Ishikawa C., Kaziro Y. The role of guanosine triphosphate in translocation reaction catalyzed by elongation factor G. J Biol Chem. 1974 Jul 10;249(13):4321–4323. [PubMed] [Google Scholar]
  13. Iwaya M., Denhardt D. T. The mechanism of replication of phi X174 single-stranded DNA. II. The role of viral proteins. J Mol Biol. 1971 Apr 28;57(2):159–175. doi: 10.1016/0022-2836(71)90339-1. [DOI] [PubMed] [Google Scholar]
  14. Mannherz H. G., Goody R. S. Proteins of contractile systems. Annu Rev Biochem. 1976;45:427–465. doi: 10.1146/annurev.bi.45.070176.002235. [DOI] [PubMed] [Google Scholar]
  15. Modrich P., Lehman I. R., Wang J. C. Enzymatic joining of polynucleotides. XI. Reversal of Escherichia coli deoxyribonucleic acid ligase reaction. J Biol Chem. 1972 Oct 10;247(19):6370–6372. [PubMed] [Google Scholar]
  16. Molineux I. J., Friedman S., Gefter M. L. Purification and properties of the Escherichia coli deoxyribonucleic acid-unwinding protein. Effects on deoxyribonucleic acid synthesis in vitro. J Biol Chem. 1974 Oct 10;249(19):6090–6098. [PubMed] [Google Scholar]
  17. Olivera B. M., Bonhoeffer F. Discontinuous DNA replication in vitro. I. Two distinct size classes of intermediates. Nat New Biol. 1972 Dec 20;240(103):233–235. doi: 10.1038/newbio240233a0. [DOI] [PubMed] [Google Scholar]
  18. Richet E., Kohiyama M. Purification and characterization of a DNA-dependent ATPase from Escherichia coli. J Biol Chem. 1976 Feb 10;251(3):808–812. [PubMed] [Google Scholar]
  19. Siden E. J., Hayashi M. Role of the gene beta-product in bacteriophage phi-X174 development. J Mol Biol. 1974 Oct 15;89(1):1–16. doi: 10.1016/0022-2836(74)90159-4. [DOI] [PubMed] [Google Scholar]
  20. Sigal N., Delius H., Kornberg T., Gefter M. L., Alberts B. A DNA-unwinding protein isolated from Escherichia coli: its interaction with DNA and with DNA polymerases. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3537–3541. doi: 10.1073/pnas.69.12.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sinsheimer R. L. Bacteriophage phi-X174 and related viruses. Prog Nucleic Acid Res Mol Biol. 1968;8:115–169. [PubMed] [Google Scholar]
  22. Sumida-Yasumoto C., Yudelevich A., Hurwitz J. DNA synthesis in vitro dependent upon phiX174 replicative form I DNA. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1887–1891. doi: 10.1073/pnas.73.6.1887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang J. C. Interaction between DNA and an Escherichia coli protein omega. J Mol Biol. 1971 Feb 14;55(3):523–533. doi: 10.1016/0022-2836(71)90334-2. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Weissbach H., Ochoa S. Soluble factors required for eukaryotic protein synthesis. Annu Rev Biochem. 1976;45:191–216. doi: 10.1146/annurev.bi.45.070176.001203. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES