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 degrees 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.