PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A motif

Nucleic Acids Res. 2000 Oct 15;28(20):3910-7. doi: 10.1093/nar/28.20.3910.


The effect of mutations in the highly conserved Y-GG/A motif of B-type DNA polymerases was studied in the DNA polymerase from the hyperthermophilic euryarchaeon Thermococcus aggregans. This motif plays a critical role in the balance between the synthesis and degradation of the DNA chain. Five different mutations of the tyrosine at position 387 (Tyr387-->Phe, Tyr387-->Trp, Tyr387-->His, Tyr387-->Asn and Tyr387-->Ser) revealed that an aromatic ring system is crucial for the synthetic activity of the enzyme. Amino acids at this position lacking the ring system (Ser and Asn) led to a significant decrease in polymerase activity and to enhanced exonuclease activity, which resulted in improved enzyme fidelity. Exchange of tyrosine to phenylalanine, tryptophan or histidine led to phenotypes with wild-type-like fidelity but enhanced PCR performance that could be related to a higher velocity of polymerisation. With the help of a modelled structure of T.aggregans DNA polymerase, the biochemical data were interpreted proposing that the conformation of the flexible loop containing the Y-GG/A motif is an important factor for the equilibrium between DNA polymerisation and exonucleolysis.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Bacillus Phages / enzymology
  • Conserved Sequence / genetics
  • Crystallography, X-Ray
  • DNA / biosynthesis
  • DNA / genetics
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / genetics
  • DNA Polymerase beta / isolation & purification
  • DNA Polymerase beta / metabolism*
  • Exonucleases / chemistry
  • Exonucleases / genetics
  • Exonucleases / metabolism
  • Kinetics
  • Lac Operon / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation*
  • Polymerase Chain Reaction / methods*
  • Protein Conformation
  • Protein Engineering*
  • Sensitivity and Specificity
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Sulfolobus / enzymology
  • Thermococcus / enzymology*


  • DNA
  • DNA Polymerase beta
  • Exonucleases