Mechanisms of duplex DNA unwinding by Escherichia coli Rep helicase and a Rep-delta-2B mutant

Pre-steady state kinetic studies were undertaken to study the mechanisms by which the Escherichia coli Rep DNA helicase and a RepΔ2B mutant, in which a protein subdomain 2B was entirely deleted, unwind double-stranded DNA. Single turnover unwinding experiments performed at high DNA substrate concentration reveal a sigmoidal dependence of the unwinding amplitudes on Rep protein concentration. Multiple turnover DNA unwinding experiments in conjunction with DNA binding kinetics reveal that Rep oligomerization limits the rate of DNA unwinding under these conditions. These experiments establish that Rep oligomerization is required for its DNA helicase activity in vitro. A RepΔ2B mutant was purified to homogeneity. Sedimentation equilibrium studies show that this mutant exists as a monomer in a buffer containing 20 mM MOPS, 200 mM NaCl, 20% v/v glycerol, and pH 6.5 at 25°C. Under conditions of excess DNA substrate, this mutant binds to the DNA substrate also as a monomer and possesses helicase activity, as shown by stopped flow fluorescence single turnover unwinding experiments. However, the fraction of DNA molecules unwound under the above conditions is only ∼1%, indicating a low processivity for DNA unwinding by the RepΔ2B monomer. Taken together, these data suggest that the helicase activity of the wild-type Rep monomer is self-inhibited, and its interaction with a second monomer is required for helicase activity in vitro. These results have broad implications for the mechanisms of helicase action in general.