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

• Published in: Nucleic acids research 2000-10, Vol.28 (20), p.3910-3917
• Main Authors: Böhlke, K, Pisani, F M, Vorgias, C E, Frey, B, Sobek, H, Rossi, M, Antranikian, G
• Format: Article
• Language: English
• Subjects: 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; hyperthermophilic archaea; hyperthermophilic microorganisms; 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; Thermococcus aggregans
• ISSN: 1362-4962; 0305-1048; 1362-4962
• 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.