Increased DNA unwinding efficiency of bacteriophage T7 DNA helicase mutant protein 4A'/E348K

J Biol Chem. 1998 Apr 3;273(14):7880-7. 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacteriophage T7 / genetics
  • Bacteriophage T7 / metabolism*
  • Base Sequence
  • DNA Helicases / genetics*
  • DNA Helicases / metabolism*
  • DNA, Viral / genetics*
  • DNA, Viral / metabolism*
  • Hydrolysis
  • Kinetics
  • Molecular Sequence Data
  • Mutation*

Substances

  • DNA, Viral
  • DNA Helicases