title: Improvement of the 3'-5' exonuclease activity of Taq DNA polymerase by protein engineering in the active site.
abstract: Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method. Taq DNA polymerase has a domain at its amino terminus (residue 1 to 291) that has a 5'-3' exonuclease activity, a 3'-5' exonuclease domain in the middle (residue 292 to 423), and a domain at its C-terminus that catalyzes polymerase reactions. Taq DNA polymerase is classified into the polI family which is represented by E. coli DNA polymerase I. The three dimensional structural alignment of 3'-5' exonuclease domains from the polI family, DNA polymerases leads us to understand why Taq DNA polymerase does not carry out proof-reading in the polymerase chain reaction. Three sequence motifs, called ExoI, II, and III must be present in order to carry out proof-reading by the 3'-5' exonuclease reaction in DNA polymerization, but Taq DNA polymerase contains none of them. The key catalytic module in the 3'-5' exonuclease is two metal ions chelated by active-site carboxylic amino acids. In order to render the 3'-5' exonuclease activity in Taq DNA polymerase, a catalytic module was constructured in the active site by protein engineering. The mutant Taq DNA polymerase shows twice as much the 3'-5' exonuclease activity as that of wild-type DNA polymerase.
authors.affiliation: School of Chemical Engineering & Technology, Yeungnam University, Kyungsan, Korea.
authors.forename: Y
authors.initials: Y
authors.lastname: Park
authors.forename: H
authors.initials: H
authors.lastname: Choi
authors.forename: D S
authors.initials: DS
authors.lastname: Lee
authors.forename: Y
authors.initials: Y
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology / Q000235:genetics
chemical_list: D019914:Taq Polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V