PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A motif.
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.
Böhlke
K
K
Institute of Technical Microbiology, Technical University Hamburg-Harburg, Denickestrabetae 15, 21073 Hamburg, Germany.
Pisani
F M
FM
Vorgias
C E
CE
Frey
B
B
Sobek
H
H
Rossi
M
M
Antranikian
G
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D017103:Bacillus Phages / Q000201:enzymology; D017124:Conserved Sequence / Q000235:genetics; D018360:Crystallography, X-Ray; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005092:Exonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007763:Lac Operon / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011487:Protein Conformation; D015202:Protein Engineering; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology; D019712:Thermococcus / Q000201:enzymology*
D004247:DNA; D019951:DNA Polymerase beta; D005092:Exonucleases