TY - JOUR
TI - PCR performance of the B-type DNA polymerase from the thermophilic
euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A
motif
AU - Bohlke, K
AU - Pisani, FM
AU - Vorgias, CE
AU - Frey, B
AU - Sobek, H and
AU - Rossi, M
AU - Antranikian, G
JO - Nucleic Acids Research
PY - 2000
VL - 28
TODO - 20
SP - 3910-3917
PB - Oxford University Press
SN - 0305-1048, 1362-4962
TODO - 10.1093/nar/28.20.3910
TODO - null
TODO - 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, Tyr381–>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.
ER -