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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
. 1974 Oct;71(10):4120–4124. doi: 10.1073/pnas.71.10.4120

Conversion of ϕX174 Viral DNA to Double-Stranded Form by Purified Escherichia coli Proteins

Sue Wickner *, Jerard Hurwitz
PMCID: PMC434340  PMID: 4610569

Abstract

The E. coli proteins that catalyze the conversion of ϕX174 single-stranded DNA to duplex DNA have now been purified extensively. The reaction depends on dnaB, dnaC(D), dnaE, and dnaG gene products, DNA elongation factors I and II, E. coli DNA binding protein, and two additional E. coli proteins, replication factors X and Y. DNA synthesis by these proteins requires ϕX174 viral DNA, dNTPs, Mg+2, and ATP. The product synthesized is full-length linear ϕX174 DNA. The reaction has been resolved into two steps. The first step involves the interaction of ATP and ϕX174 DNA with dnaB and dnaC(D) gene products, E. coli DNA binding protein, and replication factors X and Y in the absence of dNTPs. Subsequent dNMP incorporation requires the addition of DNA polymerase III, DNA elongation factors I and II, dnaG gene product, and dNTPs.

Keywords: E. coli dna gene products, E. coli DNA binding protein, DNA elongation factors I and II, replication factor X and factor Y, DNA polymerase III

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Selected References

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

  1. Campbell J. L., Soll L., Richardson C. C. Isolation and partial characterization of a mutant of Escherichia coli deficient in DNA polymerase II. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2090–2094. doi: 10.1073/pnas.69.8.2090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Gefter M. L., Hirota Y., Kornberg T., Wechsler J. A., Barnoux C. Analysis of DNA polymerases II and 3 in mutants of Escherichia coli thermosensitive for DNA synthesis. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3150–3153. doi: 10.1073/pnas.68.12.3150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Hurwitz J., Wickner S. Involvement of two protein factors and ATP in in vitro DNA synthesis catalyzed by DNA polymerase 3 of Escherichia coli. Proc Natl Acad Sci U S A. 1974 Jan;71(1):6–10. doi: 10.1073/pnas.71.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hurwitz J., Wickner S., Wright M. Studies on in vitro DNA synthesis. II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerase of E. coli. Biochem Biophys Res Commun. 1973 Mar 17;51(2):257–267. doi: 10.1016/0006-291x(73)91251-5. [DOI] [PubMed] [Google Scholar]
  6. JOVIN T., CHRAMBACH A., NAUGHTON M. A. AN APPARATUS FOR PREPARATIVE TEMPERATURE-REGULATED POLYACRYLAMIDE GEL ELECTROPHORESIS. Anal Biochem. 1964 Nov;9:351–369. doi: 10.1016/0003-2697(64)90192-7. [DOI] [PubMed] [Google Scholar]
  7. Nüsslein V., Otto B., Bonhoeffer F., Schaller H. Function of DNA polymerase 3 in DNA replication. Nat New Biol. 1971 Dec 29;234(52):285–286. doi: 10.1038/newbio234285a0. [DOI] [PubMed] [Google Scholar]
  8. Schekman R., Wickner W., Westergaard O., Brutlag D., Geider K., Bertsch L. L., Kornberg A. Initiation of DNA synthesis: synthesis of phiX174 replicative form requires RNA synthesis resistant to rifampicin. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2691–2695. doi: 10.1073/pnas.69.9.2691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Wickner R. B., Wright M., Wickner S., Hurwitz J. Conversion of phiX174 and fd single-stranded DNA to replicative forms in extracts of Escherichia coli. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3233–3237. doi: 10.1073/pnas.69.11.3233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Wickner S., Berkower I., Wright M., Hurwitz J. Studies on in vitro DNA synthesis: purification of dna C gene product containing dna D activity from Escherichia coli. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2369–2373. doi: 10.1073/pnas.70.8.2369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Wickner S., Wright M., Hurwitz J. Association of DNA-dependent and -independent ribonucleoside triphosphatase activities with dnaB gene product of Escherichia coli. Proc Natl Acad Sci U S A. 1974 Mar;71(3):783–787. doi: 10.1073/pnas.71.3.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Wickner S., Wright M., Hurwitz J. Studies on in vitro DNA synthesis. Purification of the dna G gene product from Escherichia coli. Proc Natl Acad Sci U S A. 1973 May;70(5):1613–1618. doi: 10.1073/pnas.70.5.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Wickner W., Brutlag D., Schekman R., Kornberg A. RNA synthesis initiates in vitro conversion of M13 DNA to its replicative form. Proc Natl Acad Sci U S A. 1972 Apr;69(4):965–969. doi: 10.1073/pnas.69.4.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Wickner W., Kornberg A. DNA polymerase 3 star requires ATP to start synthesis on a primed DNA. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3679–3683. doi: 10.1073/pnas.70.12.3679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wickner W., Schekman R., Geider K., Kornberg A. A new form of DNA polymerase 3 and a copolymerase replicate a long, single-stranded primer-template. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1764–1767. doi: 10.1073/pnas.70.6.1764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wright M., Wickner S., Hurwitz J. Studies on in vitro DNA synthesis. Isolation of DNA B gene product from Escherichia coli. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3120–3124. doi: 10.1073/pnas.70.11.3120. [DOI] [PMC free article] [PubMed] [Google Scholar]

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