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Nucleic Acids Research, 2000, Vol. 28, No. 20 3910-3917
© 2000 Oxford University Press

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

Kristina Böhlke, Francesca M. Pisani1, Constantinos E. Vorgias2, Bruno Frey3, Harald Sobek3, Mosè Rossi1 and Garabed Antranikian*

Institute of Technical Microbiology, Technical University Hamburg-Harburg, Denickestraße 15, 21073 Hamburg, Germany, 1Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via G. Marconi 10, 80125 Napoli, Italy, 2National and Kapodistrian University of Athens, Faculty of Biology, Department of Biochemistry–Molecular Biology, Panepistimiopolis-Kouponia, 15701 Athens, Greece and 3Roche Diagnostics GmbH, Department of Molecular Biology, Nonnenwald 2, 82377 Penzberg, Germany

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.

* To whom correspondence should be addressed. Tel: +49 40 42878 3117; Fax: +49 40 42878 2582; Email: antranikian@tu-harburg.de


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