Multi‐step DNA cleavage in rat liver nuclei is inhibited by thiol reactive agents

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Abstract
DNA fragmentation in isolated rat liver nuclei is a Mg2+-dependent, multi-step process which is potentiated by Ca2+ and cleaves the DNA into ≥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, ≥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