Abstract
Analysis by electron microscopy of the replicative intermediates synthesized in vitro in the ϕ29 replication system indicated that initiation of replication can occur from both DNA ends in the same template molecule. Type II molecules (DNA partially double-stranded and partially single-stranded) are produced by separation of the two displacement forks of double-stranded DNA molecules with two single-stranched branches when they meet.
In addition to the natural template ϕ29 DNA-terminal protein, the ϕ29 DNA polymerase is able to replicate single-stranded DNA with the sequences corresponding to the right 3’ end of ϕ29 DNA by a protein-priming mechanism. In both cases, there was a strong preference for the formation of a covalent complex between the terminal protein p3 and dAMP. However, when poly dC was used as template, p3-dGMP was preferentially formed, indicating that the template specificity for the initiation reaction is provided by the 3’ end of the template strand. In the absence of DNA, the ϕ29 DNA polymerase was able to deoxynucleotidylylate the terminal protein with any of the four dNTPs. In these conditions, the efficiency of the reaction is low, due to a strong decrease in affinity for the dNTPs. The ϕ29 DNA polymerase also catalyzed the +1 nucleotide addition on double-stranded DNA. The different dNMPs could act as template in this +1 addition reaction, as well as the 3’ end of single-stranded DNA. The 3’→5’ exonuclease activity of the ϕ29 DNA polymerase contributes to the fidelity of DNA synthesis by a factor of, at least, 100-fold. Six conserved regions of amino acid homology have been found at the carboxyl-part and in the same linear arrangement in 27 DNA-dependent DNA polymerases belonging to the two main superfamilies: pol I-like and α-like DNA polymerases. According to the three-dimensional model of the Klenow fragment these regions are located in the proposed polymerization domain. Site-directed mutagenesis in three of these regions in the ϕ29 DNA polymerase supports this model.
The nature of the sequences recognized by the viral protein p6 at the ϕ29 DNA ends, and the structure of the protein p6-DNA complex have been further studied. The fact that stimulation of the initiation reaction by protein p6 is higher at conditions that stabilize the double-helix suggests that the nucleoprotein structure formed at the ϕ29 DNA ends facilitates unwinding at the replication origin allowing initiation to occur. The ϕ29 SSB protein p5 stimulates in vitro ϕ29 DNA replication by allowing reinitiations on new templates to take place.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
- Bernad A, Blanco L, Lázaro JM, Martín G, Salas M (1989). A conserved 3’→5’ exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell 59: 219–228
- Bernad A, Lázaro JM, Salas M, Blanco L (1990). The highly conserved amino acid sequence motif 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
- Bernad A, Zaballos A, Salas M, Blanco L (1987). Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases. EMBO J. 6:4221–4225.
- Blanco L, Bernad A, Blanco MA, Salas M (1991). A general structure for DNA-dependent DNA polymerases. Gene, in press
- Blanco L, Bernad A, Lázaro JM, Martín G, Garmendia C, Salas M (1989). Highly efficient DNA systhesis by the phage ϕ29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264: 8935–8940
- Blanco L, Bernad A, Salas M (1988). Transition from initiation to elongation in protein-primed ϕ29 DNA replication: salt-dependent stimulation by the viral protein p6. J. Virol. 62: 4167–4172
- Blanco L, Prieto I, Gutiérrez J, Bernad A, Lâzaro 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
- Blanco L, Salas M (1984). Characterization and purification of a phage ϕ29-encoded DNA polymerase required for the initiation of replication. Proc. Natl. Acad. Sci. USA 81: 5325–5329
- Blanco L, Salas M (1985). Replication of phage ϕ29 DNA with purified terminal protein and DNA polymerase: Synthesis of full-length ϕ29 DNA. Proc. Natl. Acad. Sci. USA 82: 6404–6408
- 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., in press
- Clark JM (1988). Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases. Nucl. Acids. Res 16:9677–9686.
- Derbyshire V, Freemont PS, Sanderson MR, Beese L, 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
- Escarmis C, Salas M (1981). Nucleotide sequence at the termini of the DNA of Bacillus subtilis phage ϕ29. Proc. Natl. Acad. Sci. USA 78: 1446–1450
- Garmendia C, Hermoso JM, Salas M (1990). Functional domain for priming activity in the phage ϕ29 terminal protein. Gene 88: 73–79
- Garmendia C, Salas M, Hermoso JM (1988). Site-directed mutagenesis in the DNA linking site of bacteriophage ϕ29 terminal protein: isolation and characterization of a Ser 232 → Thr mutant. Nucl. Acids Res. 16: 5727–5740
- Gutiérrez C, Martin G, Sogo JM, Salas M (1991). Mechanism of stimulation of DNA replication by bacteriophage ϕ29 SSB protein p5. J. Biol. Chem. 266: 2104–2111.
- Hermoso JM, Méndez E, Soriano F, Salas M (1985). Location of the serine residue involved in the linkage between the terminal protein and the DNA of $29. Nucl. Acids. Res. 13: 7715–7728
- Inciarte MR, Salas M, Sogo JM (1980). Structure of replicating DNA molecules of Bacillus subtilis bacteriophage ϕ29. J. Virol. 34: 187–199
- Martín G, Lázaro JM, Méndez E, Salas M (1989). Characterization of the phage ϕ29 protein p5 as a single-stranded DNA binding protein. Function in ϕ29 DNA replication. Nucl. Acids. Res. 17: 3663–3672
- Mellado RP, Peôalva MA Inciarte MR, Salas M (1980). The protein covalenrly linked to the 5’ termini of the DNA of Bacillus subtilis phage ϕ29 is involved in the initiation of DNA replication. Virology 104: 84–96
- Pakula T, Caldentey J, Serrano M, Gutierrez C, Hermoso JM, Salas M, Bamford D (1990). Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication. Nucl. Acids. Res. 18: 6553–6557
- Pastrana R, Lázaro JM, Blanco L, Garda JA, Méndez E, Salas M (1985). Overproduction and purification of protein p6 of Bacillus subtilis phage ϕ29: Role in the initiation of DNA replication. Nucl. Acids Res. 13: 3083–3100
- Prieto I, Serrano M, Lâzaro JM, Salas M Hermoso JM (1988). Interaction of the bacteriophage ϕ29 protein p6 with double-stranded DNA. Proc. Natl. Acad. Sci. USA 85: 314–318
- Salas M, Mellado RP, Vinuela E, Sogo JM (1978). Characterization of a protein covalently linked to the 5’ termini of the DNA of Bacillus subtilis phage ϕ29. J. Mol. Biol. 119: 269–291
- Serrano M, Gutiérrez J, Prieto I, Hermoso JM, Salas M (1989). Signals at the bacteriophage ϕ29 DNA replication origins required for protein p6 binding and activity. EMBO J. 8:1879–1885
- Serrano M, Salas M, Hermoso JM (1990). A novel nucleoprotein complex at a replication origin. Science 248:1012–1016
- Vlcek V, Paces V (1986). Nucleotide sequence of the late region of Bacillus phage ϕ29 completes the 19285-bp sequence of ϕ29 genome. Comparison with the homologous sequence of phage PZA. Gene 46: 215–225
- Watabe K, Leusch M, Ito J (1984). Replication of bacteriophage ϕ29 DNA in vitro: The roles of terminal protein and DNA polymerase. Proc. Natl. Acad. Sci. USA 81:5374–5378
- Yoshikawa H, Friedmann T, Ito J (1981). Nucleotide sequences at the termini of ϕ29 DNA. Proc. Natl. Acad. Sci. USA 78: 1336–1340
- Zaballos A, Salas M (1989). Functional domains in the bacteriophage ϕ29 terminal protein for interaction with the $29 DNA polymerase and with DNA. Nucl. Acids. Res. 17:10353–10366
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1992 Springer-Verlag Berlin Heidelberg
About this paper
Cite this paper
Salas, M. et al. (1992). Protein-primed Replication of Bacteriophage Ø29 DNA. In: Hughes, P., Fanning, E., Kohiyama, M. (eds) DNA Replication: The Regulatory Mechanisms. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-76988-7_27
Download citation
DOI: https://doi.org/10.1007/978-3-642-76988-7_27
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-76990-0
Online ISBN: 978-3-642-76988-7
eBook Packages: Springer Book Archive