Action of mitochondrial DNAase I in destroying the capacity of isolated cell nuclei to form gels.
1. DNA prepared from non-gelable rat liver nuclei isolated in the presence of disrupted mitochondria at pH 6.0, has been compared with DNA obtained from gelable nuclei isolated at pH 4.0. The DNA of the non-gelable nuclei is partially depolymerized relative to the DNA of the gelable nuclei. 2. It has been found that sufficiently small quantities of crystallized DNAase I can cleave a very large part of the DNA of gelable nuclei isolated at pH 4 from the residual protein of these nuclei without causing extensive depolymerization of the DNA. At the same time the gelable nuclei are rendered non-gelable. 3. Partially purified DNAase II can also render gelable nuclei isolated at pH 4 non-gelable, and in so doing presumably also cleaves the DNA from the residual protein of the nuclei. 4. Mitochondrial DNAase I appears to be the enzyme responsible to a large extent for the cleavage of DNA from the residual protein of gelable rat liver cell nuclei with concomitant destruction of the gel-forming capability of these nuclei, when the nuclei are subjected to the action of disrupted mitochondria at pH 6.0 during the isolation procedure. 5. Mitochondrial DNAase II does not appear to exert appreciable action on nuclei during the course of isolation of the nuclei at pH 6.0 in the presence of disrupted mitochondria. 6. It is probable that DNAase I is not the sole enzyme responsible for destroying the gelability of nuclei isolated at pH 6.0 in the presence of disrupted mitochondria. Protease may be involved. 7. Sodium dodecyl sulfate at pH 6.0-6.3 cleaves the DNA of isolated gelable nuclei from the residual protein of these nuclei over a period of 2 to 3 hours. At pH 7.0-7.5, however, there is negligible cleavage over a period of 96 hours. 8. If non-gelable nuclei are isolated at pH 6.0 in the presence of disrupted mitochondria, DNA subsequently can be removed from them by the use of detergent at pH values ranging from 6.0-7.5 without the necessity of incubation in the detergent solution, since the DNA had already been detached from the residual protein by the action of the mitochondrial enzyme system during isolation of the nuclei.
D000818:Animals; D002467:Cell Nucleus; D004247:DNA / Q000494:pharmacology*; D003850:Deoxyribonuclease I; D003851:Deoxyribonucleases; D005782:Gels; D008928:Mitochondria; D051381:Rats
D005782:Gels; D004247:DNA; D003851:Deoxyribonucleases; D003850:Deoxyribonuclease I
CELL NUCLEUS; DESOXYRIBONUCLEASE/effects; MITOCHONDRIA