title: Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs.
abstract: Bacteriophage ϕ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of ϕ29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The results show that the addition of Helix-hairpin-Helix [(HhH)(2)] domains increases DNA binding of the hybrid polymerases without hindering their replication rate. In addition, the chimerical DNA polymerases display an improved and faithful multiply primed DNA amplification proficiency on both circular plasmids and genomic DNA and are unique ϕ29 DNA polymerase variants with enhanced amplification performance. The reported chimerical DNA polymerases will contribute to make ϕ29 DNA polymerase-based amplification technologies one of the most powerful tools for genomics.
authors.affiliation: Instituto de Biología Molecular Eladio Viñuela and Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. mdevega@cbm.uam.es
authors.forename: Miguel
authors.initials: M
authors.lastname: de Vega
authors.forename: José M
authors.initials: JM
authors.lastname: Lázaro
authors.forename: Mario
authors.initials: M
authors.lastname: Mencía
authors.forename: Luis
authors.initials: L
authors.lastname: Blanco
authors.forename: Margarita
authors.initials: M
authors.lastname: Salas
mesh_terms: D017103:Bacillus Phages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D004259:DNA-Directed DNA Polymerase
references.citation: Science. 1994 Jun 24;264(5167):1891-903
references.pmid: 7516580
references.citation: Nucleic Acids Res. 2003 Aug 15;31(16):4702-9
references.pmid: 12907710
references.citation: Cell. 1985 Dec;43(2 Pt 1):439-48
references.pmid: 4075400
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13510-5
references.pmid: 12368475
references.citation: Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074-8
references.pmid: 3156376
references.citation: J Biol Chem. 1989 May 25;264(15):8935-40
references.pmid: 2498321
references.citation: Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5847-51
references.pmid: 1631067
references.citation: Science. 2009 Nov 6;326(5954):858-61
references.pmid: 19892985
references.citation: Genomics. 1992 Jul;13(3):718-25
references.pmid: 1639399
references.citation: EMBO J. 2007 Jul 25;26(14):3494-505
references.pmid: 17611604
references.citation: Nucleic Acids Res. 2004 Oct 26;32(19):e145
references.pmid: 15507684
references.citation: J Mol Biol. 2000 Dec 1;304(3):289-300
references.pmid: 11090274
references.citation: Curr Opin Biotechnol. 2002 Feb;13(1):65-7
references.pmid: 11849960
references.citation: Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17332-6
references.pmid: 16286637
references.citation: J Biol Chem. 1994 Feb 4;269(5):3295-303
references.pmid: 8106368
references.citation: Nucleic Acids Res. 1990 Aug 25;18(16):4763-70
references.pmid: 2118623
references.citation: Nucleic Acids Res. 2001 Nov 15;29(22):E116
references.pmid: 11713336
references.citation: PLoS Genet. 2007 Sep;3(9):1702-8
references.pmid: 17892324
references.citation: Nature. 1993 Aug 19;364(6439):735-7
references.pmid: 8395022
references.citation: Methods Enzymol. 2001;334:179-92
references.pmid: 11398460
references.citation: Nucleic Acids Res. 2000 Jul 15;28(14):2643-50
references.pmid: 10908318
references.citation: Trends Microbiol. 2009 May;17(5):205-11
references.pmid: 19375325
references.citation: Trends Biotechnol. 2004 May;22(5):253-60
references.pmid: 15109812
references.citation: Biochem Biophys Res Commun. 2008 Oct 31;375(4):522-5
references.pmid: 18755142
references.citation: J Mol Biol. 1977 Feb 15;110(1):119-46
references.pmid: 845942
references.citation: Methods Enzymol. 1987;155:335-50
references.pmid: 3431465
references.citation: Methods Enzymol. 1995;262:42-9
references.pmid: 8594366
references.citation: Methods Enzymol. 1995;262:270-80
references.pmid: 8594353
references.citation: Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5261-6
references.pmid: 11959976
references.citation: J Biol Chem. 1994 Nov 25;269(47):30030-8
references.pmid: 7962004
references.citation: Proc Natl Acad Sci U S A. 2005 May 3;102(18):6407-12
references.pmid: 15845765
references.citation: Genome Res. 2001 Jun;11(6):1095-9
references.pmid: 11381035
references.citation: Mol Cell. 2004 Nov 19;16(4):609-18
references.pmid: 15546620
references.citation: BMC Res Notes. 2009 Mar 24;2:48
references.pmid: 19309528
references.citation: Proteins. 2006 Oct 1;65(1):231-8
references.pmid: 16881051
references.citation: J Biol Chem. 2002 May 3;277(18):16179-88
references.pmid: 11805086
references.citation: J Biosci Bioeng. 2009 Apr;107(4):471-3
references.pmid: 19332311
references.citation: Nucleic Acids Res. 2004 Feb 18;32(3):1197-207
references.pmid: 14973201