MULTISTEP DNA CLEAVAGE IN RAT-LIVER NUCLEI IS INHIBITED BY THIOL REACTIVE AGENTS

DNA fragmentation in isolated rat liver nuclei is a Mg2+-dependent, multi-step process which is potentiated by Ca2+ and cleaves the DNA into greater than or equal to 700, 200-300 and 30-50 kilobase pair (kbp) fragments, prior to internucleosomal cleavage by Ca2+/ Mg2+-dependent endonuclease(s). We now show that Cd2+, Hg2+, dichloroisocoumarin (DCI, a serine protease inhibitor) and N- ethylmaleimide (NEM) block both Mg2+ and Ca2+/Mg2+-dependent processes. Inhibition of DNA cleavage produced an increase in the size of the DNA fragments, from mono-/oligonucleosomes to 30-50, 200-300, greater than or equal to 700 kbp and finally to intact DNA. NEM and DCI inhibition was blocked by dithiothreitol, and it is proposed that a critical thiol(s) is involved in the DNA cleavage reactions which are a feature of the apoptotic process.