The mechanistic architecture of thermostable Pyrococcus furiosus family B DNA polymerase motif A and its interaction with the dNTP substrate.
Thermostable DNA polymerases isolated from archaeal organisms have not been completely characterized kinetically and require further study if we are to understand both their dNTP binding mechanism and their role within the organism. Here, we demonstrate that the thermostable family B DNA polymerase from Pyrococcus furiosus (Pfu Pol) contains sensitive determinants of both dNTP binding and replicational fidelity within the highly conserved motif A. Site-directed mutagenesis of the motif A SYLP region revealed that small shifts in side chain volume result in significant changes in the dNTP binding affinity, steady state kinetics, and fidelity of the enzyme. Mutants of Y410 show high fidelity in both misincorporation assays and forward mutation assays, but display a substantially higher K(m) than the wild type. In contrast, mutations of upstream residue L409 result in a drastically reduced affinity for the correct dNTP, a much higher efficiency of both misincorporation and mismatch extension, and substantially lower fidelity as demonstrated by a PCR-based forward mutation assay. The A408S mutant, however, displayed a significant increase in both dNTP binding affinity and fidelity. In summary, these data show that modulation of motif A can greatly shift both the steady and pre-steady state kinetics of the enzyme as well as the fidelity of Pfu Pol.
Kennedy
Edward M
EM
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Hergott
Christopher
C
Dewhurst
Stephen
S
Kim
Baek
B
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001681:Biological Assay; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020027:Pyrococcus furiosus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
D004259:DNA-Directed DNA Polymerase