A Two-Site Mechanism for ATP Hydrolysis by the Asymmetric Rep Dimer P2S As Revealed by Site-Specific Inhibition with ADP−AlF4

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Abstract
The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reactionthe asymmetric Rep dimer state, P2S, where ss DNA [dT(pT)15] is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP−AlF4. We found that when ADP−AlF4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s-1 per dimer (equivalent to wild-type P2S) was recovered. However, when the ADP−AlF4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP ⇄ E−ATP ⇄ E‘−ATP ⇄ E‘−ADP−Pi ⇄ E−ADP−Pi ⇄ E−ADP + Pi ⇄ E + ADP + Pi, with K1 = (2.0 ± 0.85) × 105 M-1, k2 = 22 ± 3.5 s-1, k-2 < 0.12 s-1, K3 = 4.0 ± 0.4 (k3 > 200 s-1), k4 = 1.2 ± 0.14 s-1, k-4 << 1.2 s-1, K5 = 1.0 ± 0.2 mM, and K6 = 80 ± 8 μM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E‘-ADP−Pi. In the context of our “subunit switching” model for Rep dimer translocation during processive DNA unwinding [Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol.263, 411−422], this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.