DNA fragmentation in permeabilized cells and nuclei. The role of (Ca2+ + Mg2+)-dependent endodeoxyribonuclease.
Permeabilized mammalian cells and isolated nuclei were used to study various aspects of DNA replication and repair. The present paper describes a progressive fragmentation of parental DNA in human lymphoblastoid cells that were permeabilized with L-alpha-lysophosphatidylcholine or with saponin and incubated at 37 degrees C in a DNA-synthesis mixture. The formation of DNA single-strand breaks (measured by alkaline elution) was linear with the time of incubation and was temperature-dependent. It was prevented by deleting Mg2+ or both Mg2+ and Ca2+ from the incubation mixture, or by the addition of EDTA. It was increased by deleting the components necessary for DNA synthesis, and by substituting Mn2+ for Mg2+ and Ca2+. DNA strand breaks also accumulated in isolated nuclei incubated in a DNA synthesis mixture, but not when Mg2+ was omitted. These results suggest that DNA fragmentation in permeabilized cells and nuclei was due to an activation of (Ca2+ + Mg2+)-dependent endodeoxyribonucleases. The integrity of template DNA needs to be ascertained when the conditions for measuring DNA synthesis in permeabilized cells or in nuclei are formulated.
Department of Medicine, Hebrew Rehabilitation Center for Aged, Beth Israel Hospital, Boston, MA.
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Kaminskas
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Li
D002460:Cell Line; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000201:enzymology*; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004706:Endodeoxyribonucleases / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans
D004706:Endodeoxyribonucleases; C029367:calcium magnesium dependent endodeoxyribonuclease