Figure 4.

Cleavage of the biotinylated UmuDK97A ASM mutant protein by a RecA–DNA–UmuD′ complex. Proteins were separated by SDS/PAGE and cleavage of the biotinylated ASM protein was detected by a chemiluminescent assay using streptavidin-alkaline phosphatase-conjugated antibodies. Less, but still significant, cleavage was observed when UmuD was used in the RecA–DNA complex instead of UmuD′ (data not shown). Addition of the RecA–DNA complex (+) is indicated (−, not added). The amount of UmuDK97A (substrate) in the reaction was 0.08 μg. The amount of UmuD′ (enzyme) in the reaction was 0.08 μg, 0.2 μg, 0.5 μg, or 1 μg, respectively (lanes from left to right).