Transient kinetic studies on the mechanisms of DNA binding, DNA unwinding, and DNA-stimulated ATPase activities by the Escherichia coli Rep helicase.

摘要

The mechanisms of DNA binding, DNA unwinding, and DNA-induced ATPase activities of the E. coli Rep helicase were studied using transient stopped-flow and chemical quenched-flow kinetic techniques. Binding of short ss DNA's to Rep occurs by a two-step mechanism where the bimolecular step is followed by an isomerization, which can dimerize with free protein to form the Rep P₂S dimer. The binding and dissociation kinetics of the monomeric Rep-ss DNA complexes depend hyperbolically on the ss DNA length. Rep's dimerization is independent of the length of ss DNA. Increases in temperature and the salt concentration destabilize the P₂S dimer. Presteady-state ATPase activities of the ss DNA ligated Rep monomer and the Rep dimer (P₂S) have also been characterized. Preliminary studies on ATP hydrolysis by the ss DNA-ligated Rep monomer suggest that ATP hydrolysis is coupled to unidirectional translocation (3′-to-5 ′) of the Rep monomer along ss DNA.; A two-site ATPase mechanism has been proposed for the Rep P₂S dimer in which both sites of the dimer are catalytically active and communicate allosterically. The first ATP binds to the DNA-free subunit and hydrolysis induces a global conformational change to form a high-energy intermediate with tightly bound ADP-P_i. Binding of the second ATP leads to the steady-state cycle, which occurs on the DNA-bound subunit of the P₂S dimer. The dependence of Rep unwinding activity on its oligomeric state has been performed using duplex DNA containing a 3′-ss-dT ₂₀ tail. The unwinding amplitude in the lag phase shows a sigmoidal dependence on the Rep concentration. In addition, the mixing of a helicase-deficient RepW250A mutant with the wild type protein stimulates the wtRep's unwinding activity, yet the mixing of an ATPase deficient mutant (RepK28I) inhibits unwinding in single turnover unwinding experiments. These results suggest that the active unwinding species of the Rep helicase is oligomeric.