Hibino Y, Sugano N
J Biochem. 1985 Dec;98(6):1583-90. doi: 10.1093/oxfordjournals.jbchem.a135427.
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.
将大鼠肝细胞核经孵育（自消化）释放出的核小体，通过蔗糖密度梯度离心进行分级分离，并用来自大肠杆菌的热变性³H-DNA作为外源底物进行核酸酶测定。随着孵育时间的增加，核酸酶活性增强，并定位于单/四聚体、六/七聚体和长链寡核小体级分中。相反，无论核小体大小如何，发现来自它们的0.35M NaCl可溶性级分的活性在比活性（dpm/核小体DNA）方面几乎相等。在缺乏核小体和/或染色质的蔗糖梯度（顶部区域）中未检测到这种核酸酶活性。当染色质用0.35M NaCl缓冲液透析，然后在含有0.35M NaCl的蔗糖梯度中分级分离时，大部分核酸酶活性溶解到上述顶部区域。在0.35M NaCl缓冲液中对单核小体级分进行凝胶过滤时，核酸酶活性在36,000道尔顿的位置洗脱。这种核酸酶在Mg²⁺存在下比天然DNA更快地切割热变性DNA，并且有能力在pBR322 DNA中产生单链切口和双链切口；换句话说，它具有内切核酸酶活性。此外，通过对细胞核自消化释放的核小体进行电泳，分离出了四类不同的单核小体。这些单核小体对热变性DNA也显示出核酸酶活性。因此，本研究表明，一种约36,000道尔顿的Mg²⁺依赖性内切核酸酶与大鼠肝细胞核中的核小体颗粒相关。
