PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutantions in the Y-GG/A motif. (Articolo in rivista)

Type
Label
  • PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutantions in the Y-GG/A motif. (Articolo in rivista) (literal)
Anno
  • 2000-01-01T00:00:00+01:00 (literal)
Alternative label
  • Bohlke K, Pisani FM, Vorgias CE, Frey B, Sobek H, Rossi M, Antranikian G. (2000)
    PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutantions in the Y-GG/A motif.
    in Nucleic acids research
    (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#autori
  • Bohlke K, Pisani FM, Vorgias CE, Frey B, Sobek H, Rossi M, Antranikian G. (literal)
Pagina inizio
  • 3910 (literal)
Pagina fine
  • 3917 (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#numeroVolume
  • 28 (literal)
Rivista
Note
  • ISI Web of Science (WOS) (literal)
Titolo
  • PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutantions in the Y-GG/A motif. (literal)
Abstract
  • 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. (literal)
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