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.
Widlak
P
P
Department of Experimental and Clinical Radiobiology, Center of Oncology, Gliwice, Poland.
Garrard
W T
WT
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002118:Calcium / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003300:Copper / Q000494:pharmacology; D006224:Cricetinae; D003851:Deoxyribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009994:Osmolar Concentration; D000075223:Poly-ADP-Ribose Binding Proteins; D011188:Potassium; D011994:Recombinant Proteins / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D002843:Chromatin; D007477:Ions; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins; D008345:Manganese; D003300:Copper; C113696:DFFB protein, human; D003851:Deoxyribonucleases; D020169:Caspases; D008274:Magnesium; D015032:Zinc; D011188:Potassium; D002118:Calcium