Abstract Mn 2+ Activates Multi Step DNA Cleavage in Rat Liver Nuclei

Salmaan H. Inayat Hussain*, Gerald A Cohen' and Kelvin Cain'

Department of Biomedical Science, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.
'MRC Toxicology Unit, Centre for Mechanisms of Human Toxicity, Hodgkin Building, PO Box 138, Lancaster Rd., Leicester, LE1 9HN, UK

(Received 7 August 19981 Accepted 6 October 1998)

Abstract.
The characteristic DNA cleavage patterns observed in apoptotic nuclei in vivo can be reproduced in isolated nuclei where it has been shown that initially the DNA is degraded into 'large fragments'of >700, 200-300 and 30 -0 kilobase pairs (kbp) by a Mg²⁺ dependent process which is potentiated by Ca²⁺ . These large fragments are subsequently cleaved to mono/ oligonucleosomes by Ca²⁺/Mg²⁺ activated endonuclease(s). In this study, Mn²⁺ alone can substitute for Mg²⁺ and Ca²⁺, thereby activating both 'large fragment' formation and internucleosomal cleavage in isolated rat liver nuclei. The effect of Mn²⁺ is concentration dependent in that low concentrations (<200 µM) of Mn²⁺ stimulate 'large fragment' formation only whereas higher concentrations also activate internucleosomal cleavage. The cleavage of DNA induced by either Mn²⁺, Mg²⁺ or Ca²⁺/Mg²⁺ was equally susceptible to inhibition by Zn²⁺ , N ethylmaleimide and 3,4 dichloroisocoumarin, a serine protease inhibitor. These results suggest that the multi step DNA cleavage is catalysed by a single endonuclease or a class of endonucleases which have multicationic binding sites.

Keywords: apoptosis, DNA cleavage, liver nuclei, Mn²⁺