Purification and characterization of a Mg2+-dependent endonuclease (AN34) from etoposide-treated human leukemia HL-60 cells undergoing apoptosis.
An important biochemical hallmark of apoptosis is the cleavage of chromatin into oligonucleosomal fragments. Here, we purified a Mg2+-dependent endonuclease from etoposide-treated HL-60 cells undergoing apoptosis. High levels of Mg2+-dependent endonuclease activity were detected in etoposide-treated HL-60 cells, and this activity increased in a time-dependent manner following etoposide treatment. Such an activity could not be detected in untreated cells or in cells treated with etoposide in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) or the serine protease inhibitor tosyl-L-phenylalanine chloromethyl ketone (TPCK). This Mg2+-dependent endonuclease was purified by a series of chromatographic procedures. The enzyme preparation showed a single major protein band with Mr 34,000, determined by SDS-PAGE. The presence of the Mr 34,000 Mg2+-dependent endonuclease was also confirmed by activity gel analysis. The enzyme required only Mg2+ for full activity. pH optimum was in the range of 6.5-7.5. This enzyme introduced single- and double-strand breaks into SV40 DNA and produced internucleosomal DNA cleavage in isolated nuclei from untreated cells. The DNA breaks were terminated with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation. We propose to designate this Mr 34,000 Mg2+-dependent endonuclease AN34 (apoptotic nuclease Mr 34,000).
Yoshida
A
A
Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Pourquier
P
P
Pommier
Y
Y
D000199:Actins / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D004249:DNA Damage; D053938:DNA Fragmentation / Q000187:drug effects; D004279:DNA, Viral / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D004720:Endonucleases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D009363:Neoplasm Proteins / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D013539:Simian virus 40 / Q000378:metabolism
D000199:Actins; D000972:Antineoplastic Agents, Phytogenic; D004279:DNA, Viral; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D005047:Etoposide; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C112751:apoptotic nuclease Mr 34,000; D003850:Deoxyribonuclease I; D005033:Ethylmaleimide