Fig. 1.

Illustration of the activity of DKAs in the plasmid-based PA-Nter assay. Recombinant PA-Nter (1 μg) was incubated with 1 μg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 in the presence of the test compounds. The endonucleolytic digestion of the plasmid was visualized by gel electrophoresis, and the amount of remaining intact plasmid was quantified. For L-742,001 and 10 (L-731,988), the IC₅₀ values were 0.5 μM and 0.8 μM, respectively. Wild-type (WT) enzyme showed complete cleavage with Mn²⁺ but was inactive when Mn²⁺ was omitted from the reaction mixture or replaced by Mg²⁺. The endonuclease activity was also completely abolished by the active site substitution K134A.