Fig 3. UmuD 8 cleaves as efficiently as wild-type UmuD; UmuD 18 does not cleave to UmuDʹ.

(A) Relative cleavage to UmuDʹ in the presence of RecA:ssDNA nucleoprotein filament. UmuD proteins at a concentration of 10 μM were used and the reaction was carried out for 1 h at 37°C. Percent cleavage product was determined as a ratio of the density of the UmuDʹ band to the total density of the uncleaved UmuD proteins and UmuDʹ protein for each reaction. (B) Comparison of the kinetics of cleavage of UmuD 8, UmuD 18, and wild-type UmuD. Reactions were carried out over 6 h. (C) Results for cleavage to UmuDʹ under alkaline conditions (pH 10) for 48 h are also represented. Results are normalized to cleavage of wild-type UmuD to form UmuDʹ. (D) Mixing equal amounts of UmuD 18 with the active site variant UmuD S60A results in cleavage. UmuD proteins at 10 μM were used and cleavage was carried out at 37°C for 1 h.