The association of a magnesium-dependent endonuclease activity with a nucleosome fraction from rat-liver nuclei.
The nucleosomes released by the incubation (autodigestion) of rat-liver nuclei were fractionated by sucrose-density gradient centrifugation, and subjected to nuclease assay with heat-denatured 3H-DNA from Escherichia coli as an exogenous substrate. With increasing incubation time, the nuclease activity was enhanced and localized in the mono/tetra-, hexa/hepta-, and long-chain oligonucleosome fractions. In contrast, independent of the nucleosome size, the activities of 0.35 M NaCl-soluble fractions from them were found to be almost equal in terms of specific activity (dpm/nucleosomal DNA). Such nuclease activity was not detected in the sucrose gradient (top region) lacking nucleosomes and/or chromatin. When the chromatin was dialyzed against a 0.35 M NaCl buffer and then fractionated in a sucrose gradient containing 0.35 M NaCl, most of the nuclease activity was solubilized into the above top region. On gel filtration of the mononucleosome fraction in the 0.35 M NaCl buffer, the nuclease activity was eluted at the position of 36,000 daltons. This nuclease cleaved heat-denatured DNA more rapidly than the native DNA in the presence of Mg2+, and had the ability to make both single-strand nicks and double-strand cuts in pBR322 DNA; in other words, it had an endonucleolytic activity. Moreover, four different classes of mononucleosomes were fractionated by electrophoresis of the nucleosomes released by autodigestion of the nuclei. These mononucleosomes also showed nuclease activity with the heat-denatured DNA. Thus, the present studies suggest that an Mg2+-dependent endonuclease of about 36,000 daltons is associated with the nucleosome particle(s) in rat-liver nuclei.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000378:metabolism; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000032:analysis*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009707:Nucleosomes / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains
D002843:Chromatin; D009707:Nucleosomes; D004247:DNA; D004706:Endodeoxyribonucleases; D008274:Magnesium