Multi-step DNA cleavage in rat liver nuclei is inhibited by thiol reactive agents.
DNA fragmentation in isolated rat liver nuclei is a Mg(2+)-dependent, multi-step process which is potentiated by Ca2+ and cleaves the DNA into > or = 700, 200-300 and 30-50 kilobase pair (kbp) fragments, prior to internucleosomal cleavage by Ca2+/Mg(2+)-dependent endonuclease(s). We now show that Cd2+, Hg2+, dichloroisocoumarin (DCI, a serine protease inhibitor) and N-ethylmaleimide (NEM) block both Mg2+ and Ca2+/Mg(2+)-dependent processes. Inhibition of DNA cleavage produced an increase in the size of the DNA fragments, from mono-/oligonucleosomes to 30-50, 200-300, > or = 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.
Cain
K
K
MRC Toxicology Unit, University of Leicester, UK.
Inayat-Hussain
S H
SH
Kokileva
L
L
Cohen
G M
GM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006868:Hydrolysis; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D004247:DNA; D008274:Magnesium; D005033:Ethylmaleimide; D002118:Calcium