Isolation and characterization of NUC70, a cytoplasmic, hematopoietic apoptotic endonuclease

Endonucleolytic DNA fragmentation is the common end point and the prevailin g indicator of apoptosis, We have identified a 70-kDa endonuclease (NUC70) that is activated in drug-induced apoptosis of human hematopoietic cells. W e purified NUC7O to homogeneity and generated a rabbit polyclonal antibody to distinguish it from previously identified nucleases. Biochemical charact erization of isolated NUC70 demonstrates that it is Ca2+/Mg2+-dependent and active over a pH range of 6-8, When incubated with isolated HeLa nuclei, N UC70 was capable of generating internucleosomal DNA fragmentation. This end onucleolytic activity was inhibited by Zn2+, aurintricarboxylic acid, N-eth ylnaleimide, spermine, and iodoacetamide. Western immunoblots using the ant i-NUC70 antibody and DNA-SDS-polyacrylamide gel electrophoresis assays indi cate that NUC70 expression and activity is restricted to human hematopoieti c cells. No such activity was detected in human epithelial cell lines or mu rine hematopoietic cells. We also observed no difference in levels of NUC70 expression between apoptotic and nonapoptotic cells, suggesting that activ ation of NUC70 may be by posttranslational modification. We demonstrate tha t NUC70 activity is diminished in cells pretreated with the caspase inhibit ors z-DEVD-fmk, z-VAD-fmk, and Z-CH2-Asp-DCB. Time course studies of cytopl asmic and nuclear endonuclease activities during apoptosis show that NUC70 is a cytoplasmic endonuclease that is translocated to the nucleus after the initiation of apoptosis, We confirmed this with immuno staining studies us ing anti-NUC70 antibody. These results demonstrate that NUC70 is an endogen ous cytoplasmic endonuclease that is activated during apoptosis in a caspas e-dependent mechanism.