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Bacteriophage ϕ29 DNA Polymerase

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Nucleic Acids and Molecular Biology

Part of the book series: Nucleic Acids and Molecular Biology ((NUCLEIC,volume 9))

Abstract

The most peculiar characteristic of the linear DNA molecule (19285 bp) of bacteriophage ϕ29 is the presence of a terminal protein (TP) covalently bound to both 5′ ends. The elucidation of the mechanism of ϕ29 DNA replication, together with the characterization of the proteins involved in the different stages of this process, indicated that ϕ29 TP acts as a protein primer for the initial step of ϕ29 DNA replication (reviewed in Salas 1991). Thus, after formation of a TP-dAMP complex at both DNA ends, elongation proceeds by a strand displacement mechanism to complete replication of both ϕ29 DNA strands. As will be described in this chapter, the enzymology of ϕ29 DNA replication is mainly based on the multiple catalytic activities and peculiar properties of the ϕ29 gene 2 product: the ϕ29 DNA polymerase. This enzyme, with a molecular weight of only about 66 kDa, is the only polymerase involved in ϕ29 DNA replication, catalyzing both the initiation and elongation stages of DNA synthesis (Blanco and Salas 1984, 1985a). Moreover, the peculiar polymerization properties of ϕ29 DNA polymerase (high processivity and strand displacement) make the participation of other enzymatic activities or accessory functions unnecessary to improve its efficiency in DNA synthesis (Blanco et al. 1989). From the enzymatic point of view, ϕ29 DNA polymerase is able to catalyze two distinguishable synthetic reactions: TP-deoxynucleotidylation and DNA polymerization, and also two degradative reactions: pyrophosphorolysis and 3′–5′ exonucleolysis. These multiple catalytic activities, their associated properties (summarized in Table 1), and their structural mapping, will be described in the following sections.

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References

  • Beese LS, Steitz TA (1991) Structural basis for the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J 10:25–33

    PubMed  CAS  Google Scholar 

  • Beese LS, Derbyshire V, Steitz TA (1993a) Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260:352–355

    Article  PubMed  CAS  Google Scholar 

  • Beese LS, Friedman JM, Steitz T.A. (1993b) Crystal structures of the Klenow fragment of DNA Polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate. Biochemistry 32:14095–14101

    Article  PubMed  CAS  Google Scholar 

  • Bernad A, Zaballos A, Salas M, Blanco L (1987) Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases. EMBO J 6:4219–4225

    PubMed  CAS  Google Scholar 

  • Bernad A, Blanco L, Lázaro JL, Martín G, Salas M (1989) A conserved 3′-5′ exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell 59:219–228

    Article  PubMed  CAS  Google Scholar 

  • Bernad A, Lázaro JM, Salas M, Blanco L (1990) The highly conserved amino acid sequence Tyr-Gly-Asp-Thr-Asp-Ser in α-like DNA polymerases is required by phage ϕ29 DNA polymerase for protein-primed initiation and polymerization. Proc Natl Acad Sci USA 87:4610–4614

    Article  PubMed  CAS  Google Scholar 

  • Blanco L, Salas M (1984) Characterization and purification of a phage-encoded DNA polymerase required for the initiation of replication. Proc Natl Acad Sci USA 81:5325–5329

    Article  PubMed  CAS  Google Scholar 

  • Blanco L, Salas M (1985a) Replication of phage ϕ29 DNA with purified terminal protein and DNA polymerase: synthesis of full length DNA. Proc Natl Acad Sci USA 82:6404–6408

    Article  PubMed  CAS  Google Scholar 

  • Blanco L, Salas M (1985b) Characterization of a 3′-5′ exonuclease activity in the phage ϕ29 DNA polymerase. Nucleic Acids Res 13:1239–1249

    Article  PubMed  CAS  Google Scholar 

  • Blanco L, Salas M (1995) Mutational analysis of ϕ29 DNA polymerase. Methods Enzymol (in press)

    Google Scholar 

  • Blanco L, Prieto I, Gutiérrez J, Bernad A, Lázaro JM, Hermoso JM, Salas M (1987) Effect of NH4+ ions on ϕ29 DNA-protein p3 replication: formation of a complex between the terminal protein and the DNA polymerase. J Virol 61:3983–3991

    PubMed  CAS  Google Scholar 

  • Blanco L, Bernad A, Lázaro JM, Martín G, Garmendia C, Salas M (1989) Highly efficient DNA synthesis by the phage ϕ29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem 264:8935–8940

    PubMed  CAS  Google Scholar 

  • Blanco L, Bernad A, Blasco MA, Salas M (1991) A general structure for DNA-dependent DNA polymerases. Gene 100:27–38

    Article  PubMed  CAS  Google Scholar 

  • Blanco L, Bernad A, Esteban JA, Salas M (1992a) DNA-independent deoxynucleotidylation of the ϕ29 terminal protein by the ϕ29 DNA polymerase. J Biol Chem 267:1225–1230

    PubMed  CAS  Google Scholar 

  • Blanco L, Bernad A, Salas M (1992b) Evidence favoring the hypothesis of a conserved 3′–5′ exonuclease active site in DNA-dependent DNA polymerases. Gene 112:139–144

    Article  PubMed  CAS  Google Scholar 

  • Blasco MA, Bernad A, Blanco L, Salas M (1991) Characterization and mapping of the pyrophosphorolytic activity of the phage ϕ29 DNA polymerase. Involvement of amino acid motifs highly conserved in α-like DNA polymerases. J Biol Chem 266:2904–7909

    Google Scholar 

  • Blasco MA, Esteban JA, Méndez J, Blanco L, Salas M (1992a) Structural and functional studies on ϕ29 DNA polymerase. Chromosoma 102:32–38

    Article  Google Scholar 

  • Blasco MA, Lázaro JM, Bernad A, Blanco L, Salas M (1992b) ϕ29 DNA polymerase active site: mutants in conserved residues Tyr254 and Tyr390 are affected in dNTP binding. J Biol Chem 267:19427–19434

    PubMed  CAS  Google Scholar 

  • Blasco MA, Lázaro JM, Blanco L, Salas M (1993a) ϕ29 DNA polymerase active site. The conserved amino acid motif “Kx3NSxYG” is involved in template-primer binding and dNTP selection. J Biol Chem 268:16763–16770

    PubMed  CAS  Google Scholar 

  • Blasco MA, Lázaro JM, Blanco L, Salas M (1993b) ϕ29 DNA polymerase active site. Residue Asp249 of conserved amino acid motif Dx2SLYP is critical for synthetic activities. J Biol Chem 268:24106–24113

    PubMed  CAS  Google Scholar 

  • Derbyshire V, Freemont PS, Sanderson MR, Beese LS, Friedman JM, Joyce CM, Steitz TA (1988) Genetic and crystallographic studies of the 3′, 5′-exonucleolytic site of DNA polymerase I. Science 240:199–201

    Article  PubMed  CAS  Google Scholar 

  • Derbyshire V, Grindley NDF, Joyce CM (1991) The 3′-5′ exonuclease of DNA Polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. EMBO J 10:17–24

    PubMed  CAS  Google Scholar 

  • Esteban JA, Bernad A, Salas M, Blanco L (1992) Metal activation of synthetic and degradative activities of ϕ29 DNA polymerase, a model enzyme for protein-primed DNA replication. Biochemistry 31:350–359

    Article  PubMed  CAS  Google Scholar 

  • Esteban JA, Salas M, Blanco L (1994) Fidelity of ϕ29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization. J Biol Chem 268:2719–2726

    Google Scholar 

  • Esteban JA, Soengas MS, Salas M, Blanco L (1993) 3′-5′ Exonuclease Active Site of ϕ19 DNA polymerase. Evidence favoring metal ion-assited reaction mechanism. J Biol Chem 269:31946–31954

    Google Scholar 

  • Garmendia C, Bernad A, Esteban JA, Blanco L, Salas M (1992) The bacteriophage ϕ29 DNA polymerase, a proof-reading enzyme. J Biol Chem 267:2594–2599

    PubMed  CAS  Google Scholar 

  • Kohlstaedt LA, Wang J, Friedman JM, Rice PA, Steitz TA (1992) Crystal structure at 3.5 Å resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science 256:1783–1790

    Article  PubMed  CAS  Google Scholar 

  • Kornberg A, Baker TA (1991) DNA replication, 2nd edn. WH Freeman, New York

    Google Scholar 

  • Kuriyan J, O’Donnell M (1993) Sliding clamps of DNA polymerases. J Mol Biol 234:915–925

    Article  PubMed  CAS  Google Scholar 

  • Larder BA, Kemp SD, Darby G (1987) Related functional domains in virus DNA polymerases. EMBO J 6:169–175

    PubMed  CAS  Google Scholar 

  • Leegwater PAJ, Strating M, Murphy NB, Kooy RF, van der Vliet PC, Overdulve JP (1991) The Trypanosoma brucei DNA polymerase α core subunit gene is developmentally regulated and linked to a constitutively expressed open reading frame. Nucleic Acids Res 19:6441–6447

    Article  PubMed  CAS  Google Scholar 

  • Méndez J, Blanco L, Esteban JA, Bernad A, Salas M (1992) Initiation of ϕ29 DNA replication occurs at the second 3′ nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication. Proc Natl Acad Sci USA 89:9579–9583

    Article  PubMed  Google Scholar 

  • Méndez J, Blanco L, Lazaro JM, Salas M (1994) Primel-terminus stabilization at the ϕ19 DNA polymerase active site. Mutational analysis of conserved molif TX2 GR. J Biol Chem 269:30030–30038

    PubMed  Google Scholar 

  • Ollis DL, Brick R, Hamlin R, Xuong NG, Steitz TA (1985) Structure of the large fragment of Escherichia coli DNA polymerase I complexed with TMP. Nature 313:762–766

    Article  PubMed  CAS  Google Scholar 

  • Salas M (1991) Protein-priming of DNA replication. Annu Rev Biochem 60:39–71

    Article  PubMed  CAS  Google Scholar 

  • Salas M, Méndez J, Esteban JA, Serrano M, Gutierrez C, Hermoso JM, Bravo A, Soengas MS, Lázaro JM, Blasco MA, Freire R, Bernad A, Sogo JM, Blanco L (1993) Terminal protein priming of DNA replication: bacteriophage ϕ29 as a model system. In: Doerfler W, Böhm P (eds) Virus Strategies, Molecular Biology and Pathogenesis. Verlag Chemie, Weinheim, pp 3–19

    Google Scholar 

  • Soengas MS, Esteban JA, Lázaro JM, Bernad A, Blasco MA, Salas M, Blanco L (1992) Site-directed mutagenesis at the Exo III motif of ϕ29 DNA polymerase. Overlapping structural domains for the 3′–5′ exonuclease and strand-displacement activities. EMBO J 11:4227–4237

    PubMed  CAS  Google Scholar 

Download references

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Authors and Affiliations

  1. Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049, Madrid, Spain

    L. Blanco & M. Salas

Authors
  1. L. Blanco
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  2. M. Salas
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Editors and Affiliations

  1. Max-Planck-Institut für Experimentelle Medizin, Hermann-Rein-Straße 3, 37075, Göttingen, Germany

    Fritz Eckstein

  2. Biochemistry Department, University of Dundee, Dundee, DD1 4HN, UK

    David M. J. Lilley

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© 1995 Springer-Verlag Berlin Heidelberg

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Blanco, L., Salas, M. (1995). Bacteriophage ϕ29 DNA Polymerase. In: Eckstein, F., Lilley, D.M.J. (eds) Nucleic Acids and Molecular Biology. Nucleic Acids and Molecular Biology, vol 9. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-79488-9_17

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  • DOI: https://doi.org/10.1007/978-3-642-79488-9_17

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-642-79490-2

  • Online ISBN: 978-3-642-79488-9

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