title: Mutations in the palm subdomain of Twa DNA polymerase to enhance PCR efficiency and its function analysis.
abstract: Among the family B DNA polymerases, the Twa DNA polymerase from T. wiotapuensis, a hyperthermophilic archaeon, has exceedingly high fidelity. For applications in PCR, however, the enzyme is limited by its low extension rate and processivity. To resolve these weaknesses, we focused on two amino acid residues (A381 and N501) located at the palm subdomain of Twa DNA polymerase. Following replacement of these residues by site-directed mutagenesis, Twa N501R DNA polymerase showed significantly improved polymerase function compared to the wild-type enzyme in terms of processivity (3-fold), extension rate (2-fold) and PCR efficiency. Kinetic analysis using DNA as template revealed that the kcat value of the Twa N501R mutant was similar to that of wild-type, but the Km of the Twa N501R mutant was about 1.5-fold lower than that of the wild-type. These results suggest that a positive charge at residue 501 located in the forked-point does not impede catalytic activity of the polymerase domain but stabilizes interactions between the polymerase domain and the DNA template.
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea.
authors.forename: Sung Suk
authors.initials: SS
authors.lastname: Cho
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea.
authors.forename: Mi
authors.initials: M
authors.lastname: Yu
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea. Electronic address: stkwon@skku.ac.kr.
authors.forename: Suk-Tae
authors.initials: ST
authors.lastname: Kwon
mesh_terms: D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics
chemical_list: D019951:DNA Polymerase beta
keywords: Forked-point; PCR amplification; Thermococcus waiotapuensis (Twa); Twa DNA polymerase; Twa N501R DNA polymerase