Identification of a Novel 97 kDa Endonuclease Capable of Internucleosomal DNA Cleavage

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Abstract
The two steps of DNA digestion seen in apoptotic cells were recreated in nuclei isolated from 5123tc rat hepatoma cells. The initial DNA cleavage, into high molecular weight fragments (300-50 kb), was stimulated by magnesium ions alone, whereas the second step required both calcium and magnesium ions and produced the ladder of oligonucleosomes. Endonucleolytic activities involved in both steps of DNA cleavage could be separated under appropriate conditions since the magnesium-modulated activity was tightly bound to the chromatin whereas the calcium/magnesium-dependent internucleosomal cleaving activity was easily extractable with a low ionic strength buffer. This calcium/ magnesium-dependent activity was attributed to a novel 97 kDa endonuclease which was also activated by manganese and cobalt and inhibited by millimolar concentrations of zinc, consistent with the properties ascribed to the apoptotic endonuclease. Furthermore, this activity became resistant to extraction with a low salt buffer in nuclei of apoptotic cells. Isoelectrofocusing revealed that the p97 protein existed in multiple forms of different isoelectric points (pI range 4.6-5.0), indicative of its postranslational modification. The p97 enzyme was present constitutively in a variety of cultured cells and rat tissues. It was active over a broad range of pH (6-9), but it was inactivated by reducing agents. In vitro, it displayed both endo- and exonucleolytic activities, and it was capable of both single- and double-stranded DNA cleavage. Rabbit polyclonal anti-p97 antibodies were generated and used to further distinguish this protein from other known cellular nucleases, namely, DNases I and II.