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.
Kaminskas
E
E
Department of Medicine, Hebrew Rehabilitation Center for Aged, Beth Israel Hospital, Boston, MA.
Li
J C
JC
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