Biology:Φ29 DNA polymerase

Short description: Enzyme from bacteriophage ϕ29

DNA polymerase
Identifiers
Organism	Bacillus phage phi29
Symbol	2
UniProt	P03680
Search for Structures Swiss-model Domains InterPro

DNA polymerase type B, organellar and viral, phi29-like
Identifiers
Symbol	DNA_pol_B_2
Pfam	PF03175
InterPro	IPR014416
SCOP2	2py5 / SCOPe / SUPFAM
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Φ29 DNA polymerase is an enzyme from the bacteriophage Φ29. It is being increasingly used in molecular biology for multiple displacement DNA amplification procedures, and has a number of features that make it particularly suitable for this application. It was discovered and characterized by Spanish scientists Luis Blanco and Margarita Salas.

Φ29 DNA replication

Φ29 is a bacteriophage of Bacillus subtilis with a sequenced, linear, 19,285 base pair DNA genome. Each 5' end is covalently linked to a terminal protein, which is essential in the replication process by acting as a primer for the viral DNA polymerase. A symmetrical mode of replication has been suggested, whereby protein-primed initiation occurs non-simultaneously from either end of the chromosome; this involves two replication origins and two distinct polymerase monomers. Synthesis is continual and involves a strand displacement mechanism. This was demonstrated by the ability of the enzyme to continue to copy the singly primed circular genome of the M13 phage more than tenfold in a single strand (over 70kb in a single strand). In vitro experiments have shown that Φ29 replication can proceed to completion with the sole phage protein requirements of the polymerase and the terminal protein. The polymerase catalyses the formation of the initiation complex between the terminal protein and the chromosome ends at an adenine residue. From here, continual synthesis can occur.

The polymerase

The polymerase is a monomeric protein with two distinct functional domains. Site-directed mutagenesis experiments support the proposition that this protein displays a structural and functional similarity to the Klenow fragment of the Escherichia coli Polymerase I enzyme; it comprises a C-terminal polymerase domain and a spatially separated N-terminal domain with a 3'-5' exonuclease activity.

The isolated enzyme has no intrinsic helicase activity but may carry out an equivalent function by way of its strong binding to single stranded DNA, particularly in preference to double stranded nucleic acid. This is the property of this enzyme that makes is favorably applicable to Multiple Displacement Amplification. The enzyme facilitates the "debranching" of double stranded DNA. Deoxyribonucleoside triphosphate cleavage that occurs as part of the polymerization process probably supplies the energy required for this unwinding mechanism. The continuous nature of strand synthesis (compared to the asymmetric synthesis seen in other organisms) probably contributes to this enhanced processivity. Proofreading activity conferred by the exonuclease domain was demonstrated by showing the preferential excision of a mismatched nucleotide from the 3' terminus of the newly synthesized strand. The exonuclease activity of the enzyme is, like its polymerization activity, highly processive and can degrade single-stranded oligonucleotides without dissociation. Co-operation or a 'delicate competition' between these two functional domains is essential, so as to ensure accurate elongation at an optimal rate. The exonuclease activity of the enzyme does impede its polymerization capacity; inactivation of the exonuclease activity by site-directed mutagenesis meant that a 350 fold lower dNTP concentration was required to achieve the same rates of primer elongation seen in the wild type enzyme.

Whole genome amplification

Φ29 polymerase enzyme is already used in multiple displacement amplification (MDA) procedures (including in a number of commercial kits) whereby fragments tens of kilobases in length can be produced from non-specific hexameric primers annealing at intervals along the genome. The enzyme has many desirable properties that make it appropriate for whole genome amplification (WGA) by this method.

• High processivity.
• Proofreading activity. It is believed to be 1 or 2 orders of magnitude less error prone than Taq polymerase.
• Generates large fragments, over 10kb.
• Produces more DNA than PCR-based methods, by about an order of magnitude.
• Requires minimal amount of template; 10 ng suffices.
• Novel replication mechanism; multiple-strand displacement amplification.
  • Random primers (hexamers) can be used, no need to design specific primers/target specific regions.
  • No need for thermal cycling.
• Good coverage and a reduced amplification bias when compared to PCR-based approaches. There is speculation that it is the least biased of the WGA methods in use.

Further reading

• "Identification of efficient fluorophores for the direct labeling of DNA via rolling circle amplification (RCA) polymerase φ29.". Eur J Med Chem 45 (12): 5561–6. 2010. doi:10.1016/j.ejmech.2010.09.005. PMID 20926164. 
• "Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs.". Proc Natl Acad Sci U S A 107 (38): 16506–11. 2010. doi:10.1073/pnas.1011428107. PMID 20823261. Bibcode: 2010PNAS..10716506D. 
• "phi29 DNA polymerase active site: role of residue Val250 as metal-dNTP complex ligand and in protein-primed initiation.". J Mol Biol 395 (2): 223–33. 2010. doi:10.1016/j.jmb.2009.10.061. PMID 19883660. 
• "Functional importance of bacteriophage phi29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3'-5' exonuclease active site.". J Mol Biol 391 (5): 797–807. 2009. doi:10.1016/j.jmb.2009.06.068. PMID 19576228. 
• "Rolling-circle amplification of viral DNA genomes using phi29 polymerase.". Trends Microbiol 17 (5): 205–11. 2009. doi:10.1016/j.tim.2009.02.004. PMID 19375325. 
• "Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA.". RNA 15 (5): 765–71. 2009. doi:10.1261/rna.1279909. PMID 19244362. 
• "Involvement of the TPR2 subdomain movement in the activities of phi29 DNA polymerase.". Nucleic Acids Res 37 (1): 193–203. 2009. doi:10.1093/nar/gkn928. PMID 19033368. 
• "A DNA nanotransport device powered by polymerase phi29.". Nano Lett 8 (11): 3870–8. 2008. doi:10.1021/nl802294d. PMID 18939810. 
• "Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymerase.". Biochem. Biophys. Res. Commun. 375 (4): 522–5. 2008. doi:10.1016/j.bbrc.2008.08.082. PMID 18755142. 
• "Improved multiple displacement amplification with phi29 DNA polymerase for genotyping of single human cells.". BioTechniques 44 (7): 879–90. 2008. doi:10.2144/000112755. PMID 18533898. 
• "The bacteriophage phi29 DNA polymerase.". IUBMB Life 60 (1): 82–5. 2008. doi:10.1002/iub.19. PMID 18379997. 
• "Whole Genome Amplification with Phi29 DNA Polymerase to Enable Genetic or Genomic Analysis of Samples of Low DNA Yield". Genomics Protocols. Methods in Molecular Biology. 439. 2008. pp. 1–18. doi:10.1007/978-1-59745-188-8_1. ISBN 978-1-58829-871-3. 
• "Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme.". RNA 14 (3): 503–13. 2008. doi:10.1261/rna.622108. PMID 18230765. 
• "Involvement of phage phi29 DNA polymerase and terminal protein subdomains in conferring specificity during initiation of protein-primed DNA replication.". Nucleic Acids Res 35 (21): 7061–73. 2007. doi:10.1093/nar/gkm749. PMID 17913744. 
• "Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases.". EMBO J 26 (14): 3494–505. 2007. doi:10.1038/sj.emboj.7601780. PMID 17611604. 
• "Application of Phi29 DNA polymerase in identification and full-length clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf curl Thailand virus.". Arch Virol 152 (5): 941–54. 2007. doi:10.1007/s00705-006-0914-9. PMID 17226067. 
• "Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes.". J Virol Methods 140 (1–2): 100–5. 2007. doi:10.1016/j.jviromet.2006.11.004. PMID 17174409. 
• "Repeated GenomiPhi, phi29 DNA polymerase-based rolling circle amplification, is useful for generation of large amounts of plasmid DNA.". Nucleic Acids Symp Ser (Oxf) 48 (48): 147–8. 2004. doi:10.1093/nass/48.1.147. PMID 17150521. 
• "Involvement of phi29 DNA polymerase thumb subdomain in the proper coordination of synthesis and degradation during DNA replication.". Nucleic Acids Res 34 (10): 3107–15. 2006. doi:10.1093/nar/gkl402. PMID 16757576. 
• "The phi29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition.". EMBO J 25 (6): 1335–43. 2006. doi:10.1038/sj.emboj.7601027. PMID 16511564. 
• "Cell-free cloning using phi29 DNA polymerase.". Proc Natl Acad Sci U S A 102 (48): 17332–6. 2005. doi:10.1073/pnas.0508809102. PMID 16286637. Bibcode: 2005PNAS..10217332H. 
• "Usefulness of repeated GenomiPhi, a phi29 DNA polymerase-based rolling circle amplification kit, for generation of large amounts of plasmid DNA.". Biomol Eng 22 (4): 129–32. 2005. doi:10.1016/j.bioeng.2005.05.001. PMID 16023891. 
• "A specific subdomain in phi29 DNA polymerase confers both processivity and strand-displacement capacity.". Proc Natl Acad Sci U S A 102 (18): 6407–12. 2005. doi:10.1073/pnas.0500597102. PMID 15845765. Bibcode: 2005PNAS..102.6407R. 
• "Involvement of the "linker" region between the exonuclease and polymerization domains of phi29 DNA polymerase in DNA and TP binding.". Gene 348: 89–99. 2005. doi:10.1016/j.gene.2004.12.041. PMID 15777661. 
• "Synthesis of universal unmethylated control DNA by nested whole genome amplification with phi29 DNA polymerase.". Biochem. Biophys. Res. Commun. 329 (1): 219–23. 2005. doi:10.1016/j.bbrc.2005.01.088. PMID 15721296. 
• "Application of Phi29 DNA polymerase mediated whole genome amplification on single spores of arbuscular mycorrhizal (AM) fungi.". FEMS Microbiol Lett 242 (1): 65–71. 2005. doi:10.1016/j.femsle.2004.10.041. PMID 15621421. 
• "Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29.". Mol Cell 16 (4): 609–18. 2004. doi:10.1016/j.molcel.2004.10.019. PMID 15546620. 
• "Amplification of plant genomic DNA by Phi29 DNA polymerase for use in physical mapping of the hypermethylated genomic region.". Plant Cell Rep 23 (3): 144–7. 2004. doi:10.1007/s00299-004-0806-y. PMID 15168072. 
• "phi29 DNA polymerase-terminal protein interaction. Involvement of residues specifically conserved among protein-primed DNA polymerases.". J Mol Biol 337 (4): 829–41. 2004. doi:10.1016/j.jmb.2004.02.018. PMID 15033354. 
• "A simple method for cloning the complete begomovirus genome using the bacteriophage phi29 DNA polymerase.". J Virol Methods 116 (2): 209–11. 2004. doi:10.1016/j.jviromet.2003.11.015. PMID 14738990. 
• "Function of the C-terminus of phi29 DNA polymerase in DNA and terminal protein binding.". Nucleic Acids Res 32 (1): 361–70. 2004. doi:10.1093/nar/gkh184. PMID 14729920. 
• "Two positively charged residues of phi29 DNA polymerase, conserved in protein-primed DNA polymerases, are involved in stabilisation of the incoming nucleotide.". J Mol Biol 335 (2): 481–94. 2004. doi:10.1016/j.jmb.2003.10.024. PMID 14672657. 
• "phi29 DNA polymerase residue Phe128 of the highly conserved (S/T)Lx(2)h motif is required for a stable and functional interaction with the terminal protein.". J Mol Biol 325 (1): 85–97. 2003. doi:10.1016/S0022-2836(02)01130-0. PMID 12473453. 
• "TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing.". BioTechniques Suppl: 44–7. 2002. PMID 12083397. 
• "A highly conserved lysine residue in phi29 DNA polymerase is important for correct binding of the templating nucleotide during initiation of phi29 DNA replication.". J Mol Biol 318 (1): 83–96. 2002. doi:10.1016/S0022-2836(02)00022-0. PMID 12054770. 
• "A positively charged residue of phi29 DNA polymerase, highly conserved in DNA polymerases from families A and B, is involved in binding the incoming nucleotide.". Nucleic Acids Res 30 (7): 1483–92. 2002. doi:10.1093/nar/30.7.1483. PMID 11917008. 
• "Phi29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein.". Nucleic Acids Res 30 (6): 1379–86. 2002. doi:10.1093/nar/30.6.1379. PMID 11884636. 
• "Resolution of head-on collisions between the transcription machinery and bacteriophage phi29 DNA polymerase is dependent on RNA polymerase translocation.". EMBO J 18 (20): 5675–82. 1999. doi:10.1093/emboj/18.20.5675. PMID 10523310. 
• "Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase.". J Mol Biol 292 (1): 39–51. 1999. doi:10.1006/jmbi.1999.3052. PMID 10493855. 
• "A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase.". J Mol Biol 290 (1): 241–51. 1999. doi:10.1006/jmbi.1999.2900. PMID 10388570. 
• "Role of the "YxGG/A" motif of Phi29 DNA polymerase in protein-primed replication.". J Mol Biol 286 (1): 57–69. 1999. doi:10.1006/jmbi.1998.2477. PMID 9931249. 
• "phi29 DNA polymerase residue Ser122, a single-stranded DNA ligand for 3'-5' exonucleolysis, is required to interact with the terminal protein.". J Biol Chem 273 (44): 28966–77. 1998. doi:10.1074/jbc.273.44.28966. PMID 9786901. 
• "Role of the first aspartate residue of the "YxDTDS" motif of phi29 DNA polymerase as a metal ligand during both TP-primed and DNA-primed DNA synthesis.". J Mol Biol 283 (3): 633–42. 1998. doi:10.1006/jmbi.1998.2121. PMID 9784372. 
• "DNA polymerase template switching at specific sites on the phi29 genome causes the in vivo accumulation of subgenomic phi29 DNA molecules.". Mol Microbiol 29 (3): 787–98. 1998. doi:10.1046/j.1365-2958.1998.00972.x. PMID 9723918. 
• "The RGD sequence in phage phi29 terminal protein is required for interaction with phi29 DNA polymerase.". Virology 248 (1): 12–9. 1998. doi:10.1006/viro.1998.9276. PMID 9705251. 
• "Mutational analysis of phi29 DNA polymerase residues acting as ssDNA ligands for 3'-5' exonucleolysis.". J Mol Biol 279 (4): 807–22. 1998. doi:10.1006/jmbi.1998.1805. PMID 9642062. 
• "Relating structure to function in phi29 DNA polymerase.". J Biol Chem 271 (15): 8509–12. 1996. doi:10.1074/jbc.271.15.8509. PMID 8621470. 
• "Primer-terminus stabilization at the 3'-5' exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases.". EMBO J 15 (5): 1182–92. 1996. doi:10.1002/j.1460-2075.1996.tb00457.x. PMID 8605889.