Mutants affecting nucleotide recognition by T7 DNA polymerase

• Published in: Biochemistry (Easton) 1994-01, Vol.33 (49), p.14907-14917
• Main Authors: Donlin, MJ, Johnson, KA
• Format: Article
• Language: English
• Subjects: phage T7
• ISSN: 0006-2960

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 super(-)) 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 super(-)) and Y530F(exo super(-)) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower K sub(d) values for DNA binding compared to that of wild-type(exo super(-)). With Y530F(exo super(-)) 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 super(-)), 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 super(-)). 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 K sub(d) for dCTP opposite inosine was unchanged with wild-type(exo super(-)) (197 mu M) but higher with Y530F(exo super(-)) (454 mu M) and with E480D(exo super(-)) (1 mM). The K sub(d) for dITP was the same with wild-type(exo super(-)) (180 mu M) and Y530F (exo super(-)) (229 mu M), but significantly higher with E480D(exo super(-)) (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 super(-)) and Y530F(exo super(-)) 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 super(-)). Accordingly, both mutants showed slightly lower selectivity against misincorporation. Taken together, these results indicate that E480 and Y530 each contrib