The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in a reaction that is coupled to ATP binding and hydrolysis. The Rep protein is a stable monomer in the absence of DNA but dimerizes upon binding either single-stranded or duplex DNA, and the dimer appears to be the functionally active form of the Rep helicase. As a first step toward understanding how ATP binding and hydrolysis are coupled energetically to DNA unwinding, we have investigated the kinetic mechanism of

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... de binding to the Rep monomer (P) using stopped-flow techniques and the fluorescent ATP analogue, 2'(3')-O-(N-methylanthraniloyl-ATP (mantATP). The fluorescence of mantATP is enhanced upon Rep binding due to energy transfer from tryptophan. The results are consistent with the following two-step mechanism, in which the bimolecular association step is followed by a conformational change in the P-mantATP complex: P -I-mantATP -P-mantATP c* (P-mantATP)*. The following rate and equilibrium constants were determined at 4 "C in 20 mM Tris-HC1 (pH 7.5), 6 mM NaC1, 5 mM MgCl2, and 10% Similar rate and equilibrium constants are obtained with mantATPyS, whereas the apparent rate constant for mantAMPPNP binding is 15-fold lower than for mantATP and equilibrium binding is weaker (Koverdl -lo6 M-l). Rep monomer does bind mantATP in the absence of Mg2+ (Koverdl -5 x los M-l), although the four rate constants in the above reaction increase by at least 8-fold (k-1 and k-2 increase by -100and -lOOO-fold, respectively). The affinities of Mg2+ for P-mantATP and (P-mantATP)* are 10-and 1000-fold higher than those for nucleotidefree Rep monomer, indicating that the second step in the reaction is associated with a marked increase in Mg2+ affinity. The bound Mg2+ in a (P-mantATP)*-Mg2+ complex dissociates at a rate that is comparable to the rate of mantATP release. Single-tumover kinetic studies with the Rep monomer indicate a low, but significant, DNA-independent ATPase activity, with a first-order cleavage rate constant of s-l at 4 "C, which increases with temperature (Eact = 18 f 2 kcal mol-'). These results indicate that Rep is a DNA-stimulated ATPase rather than a DNA-dependent ATPase. The absence of a burst of ADP formation in a multiple ATP tumover experiment suggests that product release is not rate-limiting under these conditions. The approaches described here and in the accompanying paper (Moore & Lohman, 1994) will be useful for subsequent studies of the more complex Rep dimeric species and of other helicases. k+l k+2 k-1 k-2 (f0.5) s-'; k-2 = 0.04 (f0.005) s-'; K1 = k+l/k-1 = (3.4 f 0.8) x lo6 M-'; K2 = k+2/k-2 = 73 (f10); DNA helicases are essential enzymes that function in DNA replication, recombination, and repair to catalyze the unwinding of double-stranded DNA (ds-DNA') to yield the single-stranded DNA (ss-DNA) intermediates that are required during these processes [for reviews, see Matson and