ENDONUCLEASE ACTIVITIES ASSOCIATED WITH HIGH-MOLECULAR-WEIGHT AND INTERNUCLEOSOMAL DNA FRAGMENTATION IN APOPTOSIS

We have used pulsed-field gel electrophoresis and electron microscopy to correlate stages of DNA fragmentation with alterations of nuclear s tructure during apoptosis. DNA fragmentation occurs in two stages. The first is initiated by a previously undescribed endonucleolytic activi ty that cleaves DNA into 50- to 300-kb fragments. Electron microscopy showed that this degree of cleavage was sufficient to cause the chroma tin to undergo condensation. The second stage of fragmentation is cata lyzed by the previously described calcium-magnesium endonuclease. The enzyme activity responsible for the initial fragmentation of DNA was f ound to be distinct from that causing subsequent internucleosomal DNA cleavage based upon its cation requirements, independence of proteolys is and lack of inhibition by zinc. Both activities were found to preex ist in nuclei from thymocytes, liver, HL60, and IL2-dependent CTLL cel ls. Thus, in apoptosis DNA degradation involves two distinct endonucle olytic activities, with only the first activity being essential for ce ll death.