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DNA fragmentation in permeabilized cells and nuclei. The role of (Ca2+ + Mg2+)-dependent endodeoxyribonuclease.

ABSTRACT: 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.

SUBMITTER: Kaminskas E

PROVIDER: S-EPMC1138774 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

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Identification of a type I Ca2+/Mg2+-dependent endonuclease induced in maize cells exposed to camptothecin.

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| S-EPMC4225560 | biostudies-literature

Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments.

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| S-EPMC1147570 | biostudies-other

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