We find that the rate of dsDNA-dependent ATPase activity is biphasic, with a fast component which represents the unwinding of the d s D N A and a slow component which results from the ssDNA-dependent ATPase activity of recBCD enzyme. Comparison of the ATPase and helicase activities permits evaluation of the efficiency of A T P hydrolysis during unwinding. This efficiency can be calculated from the maximum rates of ATPase and helicase activities and is found to range between 2.0 and 3.0 A T P

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... ecules hydrolyzed per base pair of D N A unwound. The number of ATP molecules hydrolyzed per base pair unwound is not altered by temperature but does increase a t low concentrations of D N A and high concentrations of sodium chloride and magnesium acetate. The apparent K, values for the D N A and A T P substrates of recBCD enzyme dsDNA-dependent ATPase activity at 25 OC were determined to be 0.13 nM D N A molecules and 85 pM ATP, respectively. The observed k", value is approximately 45 p M A T P s-* ( p M recBCD enzyme)-'. If this rate is corrected for the measured stoichiometry of recBCD enzyme binding to dsDNA, the k", for ATPase activity corresponds to an A T P hydrolysis rate of approximately 740 A T P molecules s-l (functional recBCD complex)-' a t 25 O C . * Address correspondence to this author. I Abbreviations: ssDNA, single-stranded DNA; dsDNA, doublestranded DNA; SSB protein, E . coli ssDNA binding protein; RF, replicative form; SDS, sodium dodecyl sulfate; DTT, diithiothreitol; ATP, adenosine triphosphate; EDTA, ethylenediaminetetraacetic acid; NADH, nicotinamide adenine dinucleotide (reduced).