THE CLONING AND EXPRESSION OF HUMAN DEOXYRIBONUCLEASE II - A POSSIBLEROLE IN APOPTOSIS

We have previously implicated deoxyribonuclease II (DNase II) as an en donuclease responsible for DNA digestion during apoptosis, The fall-le ngth human cDNA has now been cloned. The cDNA contains an open reading frame of 1078 bases coding for a 40-kDa protein. This protein is 10 k Da larger than commercially supplied enzyme, which has been proteolyti cally cleaved at an internal aspartate residue. The gene is located at chromosome 19p13.2, and has no significant homology to other human pr oteins, but has >30% identity to three predicted genes in Caenorhabdit is elegans. To determine whether overexpression of DNase II induces ap optosis in Chinese hamster ovary cells, the cDNA was cotransfected wit h a plasmid encoding green fluorescent protein. Within 24 h, a signifi cant proportion of green fluorescent protein-positive cells contained condensed chromatin, whereas vector-only controls remained viable, Con sidering that DNase II is normally active only at low pH, it was surpr ising that transfection induced chromatin condensation. To confirm tha t transfection was not activating another endonuclease, cells were inc ubated with the caspase inhibitor arbonyl-Val-Ala-Asp-(O-methyl)-fluor omethylketone; this failed to inhibit chromatin condensation induced b y DNase II. These results demonstrate that DNase II acts downstream of caspase activation and that it may be activated by an as yet unknown mechanism to induce DNA digestion during apoptosis.