The major apoptotic endonuclease DFF40/CAD is a deoxyribose-specific and double-strand-specific enzyme.
DFF40/CAD endonuclease is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. The nuclease specifically introduces DNA double strand breaks into chromatin substrates. Here we performed a detailed study on the specificity of the nuclease using synthetic single-stranded and double-stranded ribo- and deoxyribo-oligonucleotides as substrates. We have found that neither single-stranded DNA, single-stranded RNA, double-stranded RNA nor RNA-DNA heteroduplexes are cleaved by the DFF40/CAD nuclease. Noteworthy, all types of oligonucleotides that are not cleaved by the nuclease inhibit cleavage of double-stranded DNA. We have also observed that in cells undergoing apoptosis in vivo neither the activation of DFF40/CAD nor oligonucleosomal chromatin fragmentation was temporally correlated with either total cellular or nuclear RNA degradation. We conclude that DFF40/CAD is exclusively specific for double-stranded DNA.
Hanus
Jakub
J
Department of Experimental and Clinical Radiobiology, Maria Sklodowska-Curie Cancer Center and Institute of Oncology, Branch in Gliwice, 15 Wybrzeze AK, 44-100, Gliwice, Poland.
Kalinowska-Herok
Magdalena
M
Widlak
Piotr
P
D017209:Apoptosis / Q000502:physiology; D004247:DNA / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D000075223:Poly-ADP-Ribose Binding Proteins; D012313:RNA / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000075223:Poly-ADP-Ribose Binding Proteins; D012313:RNA; D004247:DNA; C113696:DFFB protein, human; D003851:Deoxyribonucleases