Disassembly of genome of higher eukaryotes: pulsed-field gel electrophoretic study of initial stages of chromatin and DNA degradation in rat liver and thymus nuclei by VM-26 and selected proteases

Abstract

Nuclei were treated with VM-26, topoisomerase II inhibitor, or exogenous proteases. Initial stages of multi-step chromatin degradation were analyzed by pulsed-field gel (PFG) electrophoresis. VM-26 induced the total genome breakdown into discrete chromatin fragments containing 0.3 Mbp DNA. Treatment of nuclei with recombinant cathepsin B and chymotrypsin also revealed 0.3-Mbp DNA fragments which were mediated by endonucleolysis. Longer incubation of nuclei with chymotrypsin exhibited the appearance of 0.05-Mbp DNA fragments and their oligomers. Proteases might trigger the detachment of chromatin from nuclear matrix and contribute to the release or activation of the endonucleolytic activity leading to the initial degradation of the DNA into high-molecular weight fragments. The fragments observed correspond in size to the suggested chromatin higher-order structures. The presented data imply that the initial stages of chromatin degradation represents in reality the genome disassembly into two basic units of genome topology—0.3-Mbp DNA chromatin loop-domains which are presumably the bits of biological information, and fundamental 0.05-Mbp DNA loopsize units which might generate the functional loops of different sizes.