FIGURE 6.

MantADP binding kinetics to RecD2·dT₂₀. A, mantADP at various concentrations was mixed in the stopped flow apparatus with 0.5 μm RecD2 with (open circles) or without (filled circles) 2.5 μm dT₂₀ under the conditions of Fig. 3. Traces were fitted by single exponentials (Equation 1). The rate constants are shown as a function of concentration and the best linear fit (Equation 3) and give an association rate constant of 23.9 ± 3.6 μm⁻¹ s⁻¹ and a dissociation rate constant of 144 ± 25 s⁻¹. B, RecD2 at various concentrations was mixed against 2.5 μm mantATP in the presence of 15 μm dT₂₀. The traces were fitted by single exponentials, and the resulting observed rate constants are plotted against concentration. A linear fit (Equation 3) gives an association rate constant with dT₂₀ bound of 17.2 ± 1.3 μm⁻¹ s⁻¹ from the slope and a dissociation rate constant of 174 ± 5 s⁻¹ from the intercept. In the absence of DNA, the fit gives an association rate constant of 20.2 ± 1.2 μm⁻¹ s⁻¹ and dissociation rate constant of 102 ± 9 s⁻¹. Inset, shown is the time course of an excess of 12.5 μm RecD2 (15 μm dT₂₀) binding 2.5 μm mantATP. C, MantADP release from RecD2·dT₂₀ is shown. 12.5 μm RecD2 was premixed with 2.5 μm mantADP in the presence or absence of 15 μm dT₂₀ before being rapidly mixed against 200 μm ADP. The time course, after the apparent lag due to the dead time of the stopped flow instrument (∼2 ms), was fitted to a single exponential giving a dissociation rate constant of 240 ± 3 s⁻¹.