DNA cleavage in rat liver nuclei activated by Mg2+ or Ca2+ + Mg2+ is inhibited by a variety of structurally unrelated inhibitors.

K. Cain, S. H. Inayat-Hussain, L. Kokileva, G. M. Cohen

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Internucleosomal DNA fragmentation is often regarded as the biochemical hallmark of apoptosis and can be reproduced in vitro in rat liver nuclei. In this study we demonstrate that DNA is initially cleaved into > or = 700, 200-250, and 30-50 kilobase pair (kbp) fragments via a Mg(2+)-dependent, multistep process which can be potentiated by Ca2+. The subsequent internucleosomal cleavage requires both Ca2+ and Mg2+. Furthermore, we show that the heavy metals Cd2+ and Hg2+, dichloroisocoumarin (a general serine protease inhibitor), and N-ethyl maleimide (NEM, a specific thiol reagent) are potent inhibitors of both the Mg(2+)- and Ca2+/Mg(2+)-stimulated DNA fragmentation. In contrast, two other serine protease inhibitors, N-alpha-tosyl-L-lysine chloromethylketone and N-tosyl-L-phenylalanine chloromethylketone are weak and ineffective, respectively, as inhibitors of DNA cleavage. Increasing inhibition of DNA cleavage is accompanied by a shift in the size of the cleaved DNA fragments, which increases from mono- + oligo-nucleosomes-->30-50 kbp-->200-300 kbp--> > or = 700 kbp-->intact DNA. Dithiothreitol, a dithiol, blocks NEM and dichloroisocoumarin inhibition, and since Cd2+ and Hg2+ are also potent--SH blocking agents it is proposed that a critical thiol is involved in the cleavage of DNA into both large kbp fragments and oligonucleosomal-sized fragments.

Original languageEnglish
Pages (from-to)631-638
Number of pages8
JournalBiochemistry and cell biology = Biochimie et biologie cellulaire
Volume72
Issue number11-12
Publication statusPublished - Nov 1994
Externally publishedYes

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DNA Cleavage
Liver
Rats
Serine Proteinase Inhibitors
DNA Fragmentation
DNA
Sulfhydryl Reagents
Nucleosomes
Dithiothreitol
Heavy Metals
Phenylalanine
Sulfhydryl Compounds
Lysine
Apoptosis

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

DNA cleavage in rat liver nuclei activated by Mg2+ or Ca2+ + Mg2+ is inhibited by a variety of structurally unrelated inhibitors. / Cain, K.; Inayat-Hussain, S. H.; Kokileva, L.; Cohen, G. M.

In: Biochemistry and cell biology = Biochimie et biologie cellulaire, Vol. 72, No. 11-12, 11.1994, p. 631-638.

Research output: Contribution to journalArticle

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