Ionic and cofactor requirements for the activity of the apoptotic endonuclease DFF40/CAD

The endonuclease DFF40/CAD mediates regulated DNA fragmentation and chromatin condensation in cells undergoing apoptosis. Here we report the enzyme's co-factor requirements, and demonstrate that the ionic changes that occur in apoptotic cells maximize DFF40/CAD activity. The nuclease requires Mg²⁺, exhibits a trace of activity in the presence of Mn²⁺, is not costimulated by Ca²⁺, is inhibited by Zn²⁺ or Cu²⁺, and has high activity over a rather broad pH range (7.0-8.5). The enzyme is thermally unstable, and is rapidly inactivated at 42°C. Enzyme activity is markedly affected by ionic strength. At the optimal [K⁺] of 50-125 mM, which is in the range of the cytoplasmic [K⁺] for cells undergoing apoptosis, the activity of DFF40/CAD for naked DNA cleavage is about 100-fold higher than at 0 or 200 mM [K⁺]. Although these ranges of ionic strength do not affect DFF40 homo-oligomer formation, at higher ionic strengths the enzyme introduces single-stranded nicks into supercoiled DNA.