Figure 5.

Effects of Mg²⁺ on the Cy3 fluorescence signal of a RecBC-reference DNA complex. (a) 10 nM of DNA I (■), DNA II (●) or DNA III (◇) was pre-bound with 2.4, 2.2 or 1.3 μM RecBC, respectively, and titrated with MgCl₂ in buffer M plus 100 mM NaCl at 25°C and the corrected Cy3 fluorescence (F_i,corr) was plotted as a function of total [MgCl₂]. The same experiments were performed in buffer M plus 400 mM NaCl for DNA I (□) and DNA II (○). Solid lines are simulations using equations (13) to (15) and the best fit values of $K_{\mathrm{Mg}}^{\mathrm{BD}}$ and $K_{\mathrm{Mg}}^{\mathrm{B}}$ ($(5\pm 2)\times {10}^2{\mathrm{M}}^{-1}$, and (8 ± 3) M⁻¹ respectively). (b) Comparisons of 10 nM of DNA I pre-bound with 2.4 μM RecBC titrated with MgCl₂ (●) or CaCl₂ (×) in buffer M plus 100 mM NaCl at 25°C. F_i,corr is plotted as a function of total [MgCl₂] or [CaCl₂]. 10 nM of DNA III pre-bound with 1.3 μM RecBC was also titrated with CaCl₂ (◇) in buffer M plus 100 mM NaCl at 25°C. (c) Comparisons of 10 nM of DNA I pre-bound with either 2.4 μM RecBC (●) or 2.4 μM RecB^ΔnucC (▽) and titrated with MgCl₂ in buffer M plus 100 mM NaCl at 25°C. F_i,corr from each experiment was normalized arbitrarily to one and plotted as a function of [MgCl₂].