Mutations in the palm subdomain of Twa DNA polymerase to enhance PCR efficiency and its function analysis
- Authors
- Cho, SS[Cho, Sung Suk]; Yu, M[Yu, Mi]; Kwon, ST[Kwon, Suk-Tae]
- Issue Date
- 20-Aug-2014
- Publisher
- ELSEVIER SCIENCE BV
- Keywords
- Thermococcus waiotapuensis (Twa); Twa DNA polymerase; Twa N501R DNA polymerase; PCR amplification; Forked-point
- Citation
- JOURNAL OF BIOTECHNOLOGY, v.184, pp.39 - 46
- Indexed
- SCIE
SCOPUS
- Journal Title
- JOURNAL OF BIOTECHNOLOGY
- Volume
- 184
- Start Page
- 39
- End Page
- 46
- URI
- https://scholarworks.bwise.kr/skku/handle/2021.sw.skku/51991
- DOI
- 10.1016/j.jbiotec.2014.05.007
- ISSN
- 0168-1656
- 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 kw 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. (C) 2014 Elsevier B.V. All rights reserved.
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Collections - Biotechnology and Bioengineering > Department of Genetic Engineering > 1. Journal Articles
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