Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs.
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.
de Vega
Miguel
M
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
Lázaro
José M
JM
Mencía
Mario
M
Blanco
Luis
L
Salas
Margarita
M
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
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D004259:DNA-Directed DNA Polymerase
Science. 1994 Jun 24;264(5167):1891-903
7516580
Nucleic Acids Res. 2003 Aug 15;31(16):4702-9
12907710
Cell. 1985 Dec;43(2 Pt 1):439-48
4075400
Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13510-5
12368475
Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074-8
3156376
J Biol Chem. 1989 May 25;264(15):8935-40
2498321
Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5847-51
1631067
Science. 2009 Nov 6;326(5954):858-61
19892985
Genomics. 1992 Jul;13(3):718-25
1639399
EMBO J. 2007 Jul 25;26(14):3494-505
17611604
Nucleic Acids Res. 2004 Oct 26;32(19):e145
15507684
J Mol Biol. 2000 Dec 1;304(3):289-300
11090274
Curr Opin Biotechnol. 2002 Feb;13(1):65-7
11849960
Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17332-6
16286637
J Biol Chem. 1994 Feb 4;269(5):3295-303
8106368
Nucleic Acids Res. 1990 Aug 25;18(16):4763-70
2118623
Nucleic Acids Res. 2001 Nov 15;29(22):E116
11713336
PLoS Genet. 2007 Sep;3(9):1702-8
17892324
Nature. 1993 Aug 19;364(6439):735-7
8395022
Methods Enzymol. 2001;334:179-92
11398460
Nucleic Acids Res. 2000 Jul 15;28(14):2643-50
10908318
Trends Microbiol. 2009 May;17(5):205-11
19375325
Trends Biotechnol. 2004 May;22(5):253-60
15109812
Biochem Biophys Res Commun. 2008 Oct 31;375(4):522-5
18755142
J Mol Biol. 1977 Feb 15;110(1):119-46
845942
Methods Enzymol. 1987;155:335-50
3431465
Methods Enzymol. 1995;262:42-9
8594366
Methods Enzymol. 1995;262:270-80
8594353
Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5261-6
11959976
J Biol Chem. 1994 Nov 25;269(47):30030-8
7962004
Proc Natl Acad Sci U S A. 2005 May 3;102(18):6407-12
15845765
Genome Res. 2001 Jun;11(6):1095-9
11381035
Mol Cell. 2004 Nov 19;16(4):609-18
15546620
BMC Res Notes. 2009 Mar 24;2:48
19309528
Proteins. 2006 Oct 1;65(1):231-8
16881051
J Biol Chem. 2002 May 3;277(18):16179-88
11805086
J Biosci Bioeng. 2009 Apr;107(4):471-3
19332311
Nucleic Acids Res. 2004 Feb 18;32(3):1197-207
14973201