Role of a GAG Hinge in the Nucleotide-induced Conformational Change Governing Nucleotide Specificity by T7 DNA Polymerase

Zhinan Jin,Kenneth A Johnson

doi:10.1074/jbc.m110.156737

Zhinan Jin, Kenneth A Johnson

Open Access

https://doi.org/10.1074/jbc.m110.156737

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Abstract

A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches.

Highlights

For the past 2 decades, there has been considerable controversy regarding the role of induced fit in enzyme specificity [6, 7]
The more important discovery from this study was that the reverse rate of the conformational change was much slower than the chemistry step, and only the forward rate of the conformational change and the ground state nucleotide binding Kd dictated the specificity for correct nucleotide incorporation
Generation of the Hinge Mutants—To characterize the conformational change during nucleotide binding and incorporation, mutants were constructed in the background of a Cyslight T7 DNA polymerase constructed by Tsai et al [16] and labeled with MDCC

Summary

Role of GAG Hinge in Polymerase Specificity

The T7 DNA polymerase in the open and closed states obtained in the absence and presence of nucleotide [15]. The mutations of these glycine residues to alanine decreased the forward rate of the conformational change ϳ1000-fold. Our data suggest that the conformational change reported by the fluorophore attached to the fingers domain corresponds to the large rotation of three catalytically important helices, which contributes significantly to the specificity of correct nucleotide incorporation

EXPERIMENTAL PROCEDURES

RESULTS

Kinetics of nucleotide binding and enzyme isomerization for the hinge mutant

Fidelity of nucleotide incorporation by the hinge mutant

DISCUSSION