Isolation and characterization of NUC70, a cytoplasmic, hematopoietic apoptotic endonuclease.
Endonucleolytic DNA fragmentation is the common end point and the prevailing indicator of apoptosis. We have identified a 70-kDa endonuclease (NUC70) that is activated in drug-induced apoptosis of human hematopoietic cells. We purified NUC70 to homogeneity and generated a rabbit polyclonal antibody to distinguish it from previously identified nucleases. Biochemical characterization 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, NUC70 was capable of generating internucleosomal DNA fragmentation. This endonucleolytic activity was inhibited by Zn2+, aurintricarboxylic acid, N-ethylmaleimide, spermine, and iodoacetamide. Western immunoblots using the anti-NUC70 antibody and DNA-SDS-polyacrylamide gel electrophoresis assays indicate that NUC70 expression and activity is restricted to human hematopoietic cells. No such activity was detected in human epithelial cell lines or murine hematopoietic cells. We also observed no difference in levels of NUC70 expression between apoptotic and nonapoptotic cells, suggesting that activation of NUC70 may be by posttranslational modification. We demonstrate that NUC70 activity is diminished in cells pretreated with the caspase inhibitors z-DEVD-fmk, z-VAD-fmk, and Z-CH2-Asp-DCB. Time course studies of cytoplasmic 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 immunostaining studies using anti-NUC70 antibody. These results demonstrate that NUC70 is an endogenous cytoplasmic endonuclease that is activated during apoptosis in a caspase-dependent mechanism.
Urbano
A
A
Department of Hematology-Oncology, Tufts New England Medical Center, Boston, Massachusetts 02111, USA.
McCaffrey
R
R
Foss
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F
D000818:Animals; D000918:Antibody Specificity; D017209:Apoptosis; D002118:Calcium / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D002451:Cell Compartmentation; D019070:Cell Lineage; D003593:Cytoplasm / Q000201:enzymology*; D004720:Endonucleases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006412:Hematopoietic Stem Cells / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D014018:Tissue Distribution
D004720:Endonucleases; D020169:Caspases; D008274:Magnesium; D002118:Calcium