Mutants affecting nucleotide recognition by T7 DNA polymerase.
Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in misincorporation assays. Both E480D(exo-) and Y530F(exo-) showed saturation kinetics with the wrong nucleotide, with binding constants of 1-3 mM compared to the estimated binding affinity of 6-8 mM with wild-type(exo-). Accordingly, both mutants showed slightly lower selectivity against misincorporation. Taken together, these results indicate that E480 and Y530 each contribute to ground state nucleotide binding and suggest that the E480 may serve to specifically stabilize the incoming dTTP of A:T base pairs to compensate for the fewer hydrogen bonds compared to G:C base pairs.
Donlin
M J
MJ
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802.
Johnson
K A
KA
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D013879:Thioredoxins / Q000378:metabolism
D017931:DNA Primers; D003854:Deoxyribonucleotides; D013879:Thioredoxins; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase