FIG. 3.

Binding of wt MmeI and its mutated variants to specific DNA sequence. The 146-bp DNA fragment from pBR322 containing a single MmeI recognition site was used as a specific DNA. The nonspecific DNA was a synthetic double-stranded 27-bp oligonucleotide (1 pmol). The binding mixtures contained 10 mM Tris-HCl, pH 7.7, 20% glycerol, 50 mM KCl, 1 mM DTT, 0.1 mg/ml bovine serum albumin. A constant amount of 64 fmol of [³²P]DNA fragment carrying the MmeI site (a 146-bp DNA fragment derived from pBR322 [32]) and an increasing amount of wt MmeI (0 to 3 nM) or its mutated variants were added as indicated on the x axis. (A) DNA shifts obtained in the presence of increasing concentrations of wt MmeI (from left to right, indicated by a triangle above the picture). I, free DNA; II, MmeI-DNA complexes; III, gel wells. The obtained bands were visualized by autoradiography, and intensities of the bands were processed digitally. (B to H) Graphical representations of binding of wt MmeI and MmeI mutated variants to a 146-bp DNA fragment carrying the MmeI site. The binding of MmeI to the DNA fragment carrying the MmeI site was determined as the concentration of MmeI required for recruitment of 50% of the DNA into a DNA-protein complex(C₅₀). Curve fitting was done with the use of Sigma Plot 2000 v. 6.0.