Modulating the pKa of a Tyrosine in KlenTaq DNA Polymerase that Is Crucial for Abasic Site Bypass by in Vivo Incorporation of a Non-canonical Amino Acid

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2014
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Prokup, Alexander
Deiters, Alexander
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ChemBioChem. 2014, 15(12), pp. 1735-1737. ISSN 1439-4227. eISSN 1439-7633. Available under: doi: 10.1002/cbic.201400051
Zusammenfassung

It is estimated that about 10 000 abasic sites are formed per day per cell. Abasic sites impose a significant challenge for bypass synthesis by DNA polymerases. Recently, a tyrosine in KlenTaq DNA polymerase has been highlighted as being crucial for nucleotide selection opposite abasic sites. Structural data indicated a hydrogen bond between the tyrosine's hydroxy group and the N3 of an incoming ddATP opposite the abasic site. In order to further investigate abasic site bypass, we incorporated the unnatural amino acid 2,3,5-trifluorotyrosine at the position of the crucial tyrosine of KlenTaq DNA polymerase. Fluorine substitution at the tyrosine decreased the pka value of the tyrosine's hydroxy group and allowed its protonation state to be modulated. Single-nucleotide-incorporation experiments revealed reduced activity for the KlenTaq mutant compared to the wild-type when bypassing an abasic site analogue. The finding stresses the involvement of this tyrosine and its hydrogen bonding in abasic site bypass.

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540 Chemie
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abasic site, DNA replication, fluorotyrosine, protein engineering, unnatural amino acid mutagenesis
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ISO 690BLATTER, Nina, Alexander PROKUP, Alexander DEITERS, Andreas MARX, 2014. Modulating the pKa of a Tyrosine in KlenTaq DNA Polymerase that Is Crucial for Abasic Site Bypass by in Vivo Incorporation of a Non-canonical Amino Acid. In: ChemBioChem. 2014, 15(12), pp. 1735-1737. ISSN 1439-4227. eISSN 1439-7633. Available under: doi: 10.1002/cbic.201400051
BibTex
@article{Blatter2014-08-18Modul-28332,
  year={2014},
  doi={10.1002/cbic.201400051},
  title={Modulating the pK<sub>a</sub> of a Tyrosine in KlenTaq DNA Polymerase that Is Crucial for Abasic Site Bypass by in Vivo Incorporation of a Non-canonical Amino Acid},
  number={12},
  volume={15},
  issn={1439-4227},
  journal={ChemBioChem},
  pages={1735--1737},
  author={Blatter, Nina and Prokup, Alexander and Deiters, Alexander and Marx, Andreas}
}
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    <dcterms:abstract xml:lang="eng">It is estimated that about 10 000 abasic sites are formed per day per cell. Abasic sites impose a significant challenge for bypass synthesis by DNA polymerases. Recently, a tyrosine in KlenTaq DNA polymerase has been highlighted as being crucial for nucleotide selection opposite abasic sites. Structural data indicated a hydrogen bond between the tyrosine's hydroxy group and the N3 of an incoming ddATP opposite the abasic site. In order to further investigate abasic site bypass, we incorporated the unnatural amino acid 2,3,5-trifluorotyrosine at the position of the crucial tyrosine of KlenTaq DNA polymerase. Fluorine substitution at the tyrosine decreased the pka value of the tyrosine's hydroxy group and allowed its protonation state to be modulated. Single-nucleotide-incorporation experiments revealed reduced activity for the KlenTaq mutant compared to the wild-type when bypassing an abasic site analogue. The finding stresses the involvement of this tyrosine and its hydrogen bonding in abasic site bypass.</dcterms:abstract>
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