title: Increased DNA unwinding efficiency of bacteriophage T7 DNA helicase mutant protein 4A'/E348K.
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.
authors.affiliation: Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: M T
authors.initials: MT
authors.lastname: Washington
authors.forename: S S
authors.initials: SS
authors.lastname: Patel
mesh_terms: D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D004279:DNA, Viral; D004265:DNA Helicases