Thermodynamic and kinetic studies of the mechanism of duplex DNA unwinding by the Escherichia coli UvrD helicase

The E. coli UvrD protein is a helicase involved in DNA repair. I investigated the energetics of UvrD self assembly, both in the absence and presence of a duplex DNA substrate, using analytical ultracentrifugation methods, and have determined the subunit stoichiometry required to form the active helicase using quantitative transient kinetic methods. UvrD self-associates to form dimers and tetramers, over a range of solution conditions (from 5°C to 25°C; from 20 to 125 mM NaCl; and from 15 to 25% (v/v) glycerol; pH 8.3). In general, increasing [NaCl] and/or [glycerol] destabilizes higher order species. In a molar excess of a duplex DNA unwinding substrate, sedimentation equilibrium studies showed only one UvrD monomer bound per DNA, however, when [UvrD] is in excess over DNA, three UvrD monomers can bind. UvrD tetramers do not bind the DNA substrate, suggesting the tetramer is not a functional helicase. Transient kinetic studies, performed under single turnover conditions, showed UvrD monomers have no helicase activity, and that UvrD dimers are required for activity in vitro. A minimal kinetic mechanism for the formation of the active UvrD dimer-DNA complex was determined. The active helicase can form via two kinetic pathways. One path is the sequential binding of two monomers to the DNA substrate, while another path is the direct binding of the dimer. These experiments show that specific protein-protein contacts are required to form the functional helicase dimer. I also have shown that UvrD unwinds non-natural DNA substrates that contain a 3′ ss DNA tail within which is embedded either a polyethylene glycol spacer, or a region of ss DNA with reversed backbone polarity. These results rule out a “passive” DNA unwinding mechanism that requires the helicase to maintain continuous contact, via a single DNA binding site, with the ss DNA during the unwinding reaction. Furthermore, the functional form of UvrD must have at least two DNA binding sites, allowing the helicase to bridge the “block”, which is consistent with the determination that UvrD dimers are functional helicases. Based on these results, a detailed model is proposed to explain UvrD catalyzed DNA unwinding.