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.