Increased DNA Unwinding Efficiency of Bacteriophage T7 DNA Helicase Mutant Protein 4A′/E348K

M Todd Washington,Smita S Patel

doi:10.1074/jbc.273.14.7880

Abstract

Bacteriophage T7 4A' protein is a DNA helicase that unwinds DNA in a reaction coupled to dTTP hydrolysis. To understand better its mechanism of DNA unwinding, we characterized a set of 4A' mutant proteins (Washington, M. T., Rosenberg, A. H., Griffin, K., Studier, F. W., and Patel, S. S. (1996) J. Biol. Chem. 271, 26825-26834). We showed here, using single turnover DNA unwinding assays, that the 4A'/E348K mutant protein had the unusual property of unwinding DNA (with a 5-6-fold slower rate) despite a significant defect in its dTTPase activity (a 25-30-fold slower rate). Comparing the DNA unwinding rates to the dTTPase rates, we estimated the DNA unwinding efficiencies of both wild-type (about 1 base pair unwound per dTTP hydrolysis) and mutant (4 to 6 base pairs unwound per dTTP hydrolysis). Thus the mutant had a 4-6-fold improvement in its DNA unwinding efficiency over that of the wild-type. We believe that this mutant undergoes less slippage (uncoupled dTTP hydrolysis) than the wild-type. We speculate that nature has selected for a high rate of DNA unwinding rather than a high efficiency of DNA unwinding. Thus even though the mutant is more efficient at DNA unwinding, the wild-type probably was selected because it unwinds DNA faster.

Highlights

DNA helicases are motor proteins that catalyze processive DNA unwinding fueled by nucleoside triphosphate (NTP)1 hydrolysis [1]
We report that despite a 25–30-fold defect in DNA-stimulated deoxythymidine triphosphate (dTTP) hydrolysis activity, the mutant 4AЈ/ E348K protein surprisingly has a significant amount of DNA unwinding activity
We have examined the kinetics of dTTP hydrolysis and DNA unwinding by the wild-type 4AЈ protein and a mutant 4AЈ/ E348K protein to estimate the DNA unwinding efficiencies of both proteins

Summary

Introduction

DNA helicases are motor proteins that catalyze processive DNA unwinding fueled by nucleoside triphosphate (NTP) hydrolysis [1]. Amino acid residues in conserved motif 1, which is thought to be involved in dTTP binding and hydrolysis [12], have been changed in two sitedirected mutant proteins [7, 8]. These mutant proteins are defective in dTTP hydrolysis activity and DNA unwinding activity. Amino acid residues in conserved motif 4 have been changed in three other site-directed mutant proteins [9] These mutant proteins are defective, either directly or indirectly, in hexamer formation. Mutant proteins in one class can hydrolyze dTTP with near wild-type activity, but these proteins are defective in coupling the energy from dTTP hydrolysis to DNA unwinding. We speculate that in the case of the bacteriophage T7 4AЈ helicase protein, nature has selected in favor of a high rate of DNA unwinding and not in favor of a high efficiency of DNA unwinding

Methods

Results

Conclusion