FIG. 4.
Nitrocellulose filter-binding assays. (A to C) Binding isotherms. Purified Pol with an active concentration of 52 nM (A) or purified Pol/UL42 complexes with active concentrations of 10 nM (B) and 11 nM (C) were incubated with increasing concentrations of 32P-labeled P/T in buffer C containing either 50 mM (A and B) or 125 mM (C) KCl and applied in duplicate to a filter stack containing nitrocellulose and DEAE to trap DNA-protein complexes and free DNA, respectively. The data were fitted to equation 3 to predict the maximum stoichiometry of DNA-protein complexes and the DNA concentration at half-maximum binding (Kd). (D) Scatchard plots of the same data fitted by linear regression analysis. The negative values of the reciprocals of the slopes give estimates of P/T binding Kds of 11.5 nM for Pol (•) and 1.7 nM for Pol/UL42 (▪) in 50 mM KCl and 1.0 nM for Pol/UL42 in 125 mM KCl (▴).