Fig. 3.

Residues E47 of Int and R63 of Xis are required for Int–Xis–DNA ternary complex formation. Proteins were tested in a gel-shift assay for their respective abilities to bind to a 40-bp duplex containing the P2X1 binding sites. (A) The substrate DNA (2 nM) was preincubated at 25°C for 10 min with Xis (850 nM). Wild-type, E47A, or R30D Ints (500 nM) were then added to the first tube, followed by two serial 2-fold dilutions and the reactions incubated at 19°C for 30 min. (B) The substrate DNA (2 nM) was preincubated at 25°C for 10 min with Int (118 nM). Wild-type or R63E Xis proteins (600 nM) were added to the first tube followed by two serial 2-fold dilutions and the reactions incubated at 19°C for 30 min. For all three panels, reactions were analyzed by electrophoresis on 8% native polyacrylamide gels. The gels were then dried and visualized by autoradiography.