ATPase Activity of Escherichia coli Rep Helicase Is Dramatically Dependent on DNA Ligation and Protein Oligomeric States

Abstract

The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA using the energy derived from ATP binding and hydrolysis. Rep functions as a dimer but assembles to its active dimeric form only on binding DNA. Each protomer of a dimer contains a DNA binding site that can bind either single-stranded (S) or duplex (D) DNA. The dimer can bind up to two oligodeoxynucleotides in five DNA-ligation states: two half-ligated states, P₂S and P₂D, and three fully-ligated states, P₂S₂, P₂D₂, and P₂SD. We have previously shown that the relative stabilities of these ligation states are allosterically regulated by the binding and hydrolysis of ATP and have proposed an “active rolling” model for DNA unwinding where the enzyme cycles through a series of these ligation states in a process that is coupled to the catalytic cycle of ATP hydrolysis [Wong, I., & Lohman, T. M., (1992) Science256, 350−355]. The basal ATPase activity of Rep protein is stimulated by ss DNA binding and by protein dimerization. We have measured the steady-state ATPase activities of Rep bound to dT(pT)₁₅ in each distinct ss DNA ligation state (PS, P₂S, and P₂S₂) to compare with our previous measurements with unligated Rep monomer (P) [Moore, K. J. M., & Lohman, T. M. (1994) Biochemistry 33, 14550]. We find the ATPase activity of Rep is influenced dramatically by both dimerization and ss DNA ligation state, with the following k_cat values for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 × 10^-3 s^-1 for P, 2.17 ± 0.04 s^-1 for PS, 16.5 ± 0.2 s^-1 for P₂S, and 71 ± 2.5 s^-1 for P₂S₂ (20 mM Tris-HCl, pH 7.5, 6 mM NaCl, 5 mM MgCl₂, 10% glycerol, 4 °C). The apparent K_M's for ATP hydrolysis are 2.05 ± 0.1 μM for PS and 2.7 ± 0.2 μM for P₂S. These widely different ATPase activities reflect the allosteric effects of DNA ligation and demonstrate that cooperative communication occurs between the ATP and DNA sites of both subunits of the Rep dimer. These results further emphasize the need to explicitly consider the population distribution of oligomerization and DNA ligation states of the helicase when attempting to infer information about elementary processes such as helicase translocation based solely on macroscopic steady-state ATPase measurements.