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Mutations in the palm subdomain of Twa DNA polymerase to enhance PCR efficiency and its function analysis

Paper ID Volume ID Publish Year Pages File Format Full-Text
23186 43419 2014 8 PDF Available
Title
Mutations in the palm subdomain of Twa DNA polymerase to enhance PCR efficiency and its function analysis
Abstract

•Mutant Twa N501R DNA polymerase showed improved polymerase function compared to the wild-type enzyme in terms of processivity, extension rate and PCR efficiency.•The N501R mutant amplified a 2-kb target gene with a 10 s extension time per cycle and could amplify a 10-kb DNA fragment.•Mutant Twa N501R DNA polymerase outperformed the wild-type Twa DNA polymerase with increased amplification quantity.•A positive charge at residue 501 located in the forked-point stabilizes interactions between the polymerase domain and the DNA template.

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.

Keywords
Thermococcus waiotapuensis (Twa); Twa DNA polymerase; Twa N501R DNA polymerase; PCR amplification; Forked-point
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 184, 20 August 2014, Pages 39–46
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us