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. 1974 May;71(5):1758–1762. doi: 10.1073/pnas.71.5.1758

Synthesis of Bacteriophage Lambda DNA In Vitro: Requirement for O and P Gene Products

Hiroaki Shizuya 1,*, Charles C Richardson 1
PMCID: PMC388318  PMID: 4525790

Abstract

We have developed a cell-free system to study bacteriophage λ DNA replication. Maximal DNA synthesis in vitro requires the four deoxynucleoside triphosphates, ATP, and exogenous λ DNA. DNA synthesis requires the products of the phage O and P genes but is not inhibited by λ repressor. The kinetics of synthesis is linear for 10-15 min; however, the product of synthesis amounts to only 0.5-1% of the added template DNA. As judged by isopycnic analysis, extensive regions of the template are copied. Sedimentation analysis indicates that all of the product consists of short (11S) DNA chains. Fractions partially purified from λO+P+-infected cell extracts will complement extracts prepared from λO- or λP--infected cells.

Keywords: Escherichia coli, DNA initiation, DNA polymerase

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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 A. Sensitive mutants of bacteriophage lambda. Virology. 1961 May;14:22–32. doi: 10.1016/0042-6822(61)90128-3. [DOI] [PubMed] [Google Scholar]
  2. Ganesan A. T. Adenosine triphosphate-dependent synthesis of biologically active DNA by azide-poisoned bacteria. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1296–1300. doi: 10.1073/pnas.68.6.1296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ginsberg B., Hurwitz J. Unbiased synthesis of pulse-labeled DNA framents of bacteriophage lambda and T4. J Mol Biol. 1970 Sep 14;52(2):265–280. doi: 10.1016/0022-2836(70)90030-6. [DOI] [PubMed] [Google Scholar]
  4. Gross J. D. DNA replication in bacteria. Curr Top Microbiol Immunol. 1972;57:39–74. doi: 10.1007/978-3-642-65297-4_2. [DOI] [PubMed] [Google Scholar]
  5. Klein A., Powling A. Initiation of lambda DNA replication in vitro. Nat New Biol. 1972 Sep 20;239(90):71–73. doi: 10.1038/newbio239071a0. [DOI] [PubMed] [Google Scholar]
  6. Knippers R., Strätling W. The DNA replicating capacity of isolated E. coli cell wall-membrane complexes. Nature. 1970 May 23;226(5247):713–717. doi: 10.1038/226713a0. [DOI] [PubMed] [Google Scholar]
  7. Kuempel P. L., Veomett G. E. A possible function of DNA polymerase in chromosome replication. Biochem Biophys Res Commun. 1970 Nov 25;41(4):973–980. doi: 10.1016/0006-291x(70)90180-4. [DOI] [PubMed] [Google Scholar]
  8. Matsubara K., Kaiser A. D. Lambda dv: an autonomously replicating DNA fragment. Cold Spring Harb Symp Quant Biol. 1968;33:769–775. doi: 10.1101/sqb.1968.033.01.088. [DOI] [PubMed] [Google Scholar]
  9. Moses R. E., Richardson C. C. Replication and repair of DNA in cells of Escherichia coli treated with toluene. Proc Natl Acad Sci U S A. 1970 Oct;67(2):674–681. doi: 10.1073/pnas.67.2.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ogawa T., Tomizawa J. Replication of bacteriophage DNA. I. Replication of DNA of lambda phage defective in early functions. J Mol Biol. 1968 Dec 14;38(2):217–225. doi: 10.1016/0022-2836(68)90407-5. [DOI] [PubMed] [Google Scholar]
  11. Okazaki R., Arisawa M., Sugino A. Slow joining of newly replicated DNA chains in DNA polymerase I-deficient Escherichia coli mutants. Proc Natl Acad Sci U S A. 1971 Dec;68(12):2954–2957. doi: 10.1073/pnas.68.12.2954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Schaller H., Otto B., Nüsslein V., Huf J., Herrmann R., Bonhoeffer F. Deoxyribonucleic acid replication in vitro. J Mol Biol. 1972 Jan 28;63(2):183–200. doi: 10.1016/0022-2836(72)90369-5. [DOI] [PubMed] [Google Scholar]
  13. Schnös M., Inman R. B. Position of branch points in replicating lambda DNA. J Mol Biol. 1970 Jul 14;51(1):61–73. doi: 10.1016/0022-2836(70)90270-6. [DOI] [PubMed] [Google Scholar]
  14. Sharp P. A., Hsu M. T., Otsubo E., Davidson N. Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. I. Structure of F-prime factors. J Mol Biol. 1972 Nov 14;71(2):471–497. doi: 10.1016/0022-2836(72)90363-4. [DOI] [PubMed] [Google Scholar]
  15. Sippel A., Hartmann G. Mode of action of rafamycin on the RNA polymerase reaction. Biochim Biophys Acta. 1968 Mar 18;157(1):218–219. doi: 10.1016/0005-2787(68)90286-4. [DOI] [PubMed] [Google Scholar]
  16. Smith D. W., Schaller H. E., Bonhoeffer F. J. DNA synthesis in vitro. Nature. 1970 May 23;226(5247):711–713. doi: 10.1038/226711a0. [DOI] [PubMed] [Google Scholar]
  17. Strätling W., Ferdinand F. J., Krause E., Knippers R. Bacteriophage T7-DNA replication in vitro: an experimental system. Eur J Biochem. 1973 Sep 21;38(1):160–169. doi: 10.1111/j.1432-1033.1973.tb03046.x. [DOI] [PubMed] [Google Scholar]
  18. Sugino A., Hirose S., Okazaki R. RNA-linked nascent DNA fragments in Escherichia coli. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1863–1867. doi: 10.1073/pnas.69.7.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tomizawa J., Ogawa T. Replication of phage lambda DNA. Cold Spring Harb Symp Quant Biol. 1968;33:533–551. doi: 10.1101/sqb.1968.033.01.061. [DOI] [PubMed] [Google Scholar]
  20. Vosberg H. P., Hoffmann-Berling H. DNA synthesis in nucleotide-permeable Escherichia coli cells. I. Preparation and properties of ether-treated cells. J Mol Biol. 1971 Jun 28;58(3):739–753. doi: 10.1016/0022-2836(71)90037-4. [DOI] [PubMed] [Google Scholar]
  21. Weiss B., Live T. R., Richardson C. C. Enzymatic breakage and joining of deoxyribonucleic acid. V. End group labeling and analysis of deoxyribonucleic acid containing single straned breaks. J Biol Chem. 1968 Sep 10;243(17):4530–4542. [PubMed] [Google Scholar]
  22. 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]

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