ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: Implications for ATP driven helicase translocation

To examine the coupling of ATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a single-stranded hexadecameric oligodeoxynucleotide (S). Crosslinked Rep monomers (PS) as well as singly ligated (P₂S) and doubly ligated (P₂S₂) Rep dimers were characterized. The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P₂S crosslinked complexes were identical to the values determined for uncrosslinked Rep complexes formed with dT₁₆. Therefore, ATP hydrolysis by both PS and P₂S complexes are not coupled to DNA dissociation. This also rules out a strictly unidirectional sliding mechanism for ATP-driven translocation along single-stranded DNA by either PS or the P₂S dimer. However, ATP hydrolysis by the doubly ligated P₂S₂ Rep dimer is coupled to single-stranded DNA dissociation from one subunit of the dimer, although loosely (low efficiency). These results suggest that ATP hydrolysis can drive translocation of the dimeric Rep helicase along DNA by a "rolling" mechanism where the two DNA binding sites of the dimer alternately bind and release DNA. Such a mechanism is biologically important when one subunit binds duplex DNA, followed by subsequent unwinding.