Conconi A, Losa R, Koller T, Sogo J M
J Mol Biol. 1984 Oct 5;178(4):920-8. doi: 10.1016/0022-2836(84)90319-x.
We purified soluble rat liver chromatin and H1-depleted chromatin and photocrosslinked its DNA with psoralen at pH 7. Digestion of this chromatin with micrococcal nuclease produced a normal nucleosomal repeat. Chromatin was photoreacted in the presence of 0 to 700 mM-NaCl and was fractionated in sucrose gradients containing the same NaCl concentrations. The dissociation of H1 occurred as in the non-crosslinked controls and no preferential dissociation of core histones was observed. The samples between 100 and 500 mM-NaCl showed precipitation. In the electron microscope, the fibers appeared indistinguishable from the controls at low ionic strength. In the presence of 40 mM-NaCl, the fibers of the photoreacted chromatin were slightly more compact than the controls, and at 500 mM-NaCl, despite the complete dissociation of H1, there were still apparently intact fibers at this ionic strength. The disruption of the psoralen-treated chromatin fibers occurred only in 600 mM-NaCl, as opposed to 500 mM-NaCl in controls. The DNA of all the photoreacted samples was spread for electron microscopy under denaturing conditions. They revealed, for all the samples, single-stranded bubbles corresponding to 200 to 400 base-pairs in size. H1-depleted chromatin containing stoichiometric amounts of core histones was photoreacted at pH 10 and very low ionic strength. Under these conditions many of the nucleosomes appeared to be unraveled, although to a variable extent. In the electron microscope, the purified DNA from these samples showed extensive crosslinking when spread under denaturing conditions. These observations show that histone-DNA interactions different from those in intact nucleosomes may be created, which allow extensive access of psoralen to the DNA.
我们纯化了可溶性大鼠肝脏染色质和H1缺失染色质，并在pH 7条件下用补骨脂素对其DNA进行光交联。用微球菌核酸酶消化这种染色质产生了正常的核小体重复序列。染色质在0至700 mM - NaCl存在下进行光反应，并在含有相同NaCl浓度的蔗糖梯度中进行分级分离。H1的解离情况与非交联对照相同，未观察到核心组蛋白的优先解离。100至500 mM - NaCl之间的样品出现沉淀。在电子显微镜下，低离子强度下的纤维与对照难以区分。在40 mM - NaCl存在下，光反应染色质的纤维比对照略紧凑，在500 mM - NaCl时，尽管H1完全解离，但在此离子强度下仍有明显完整的纤维。补骨脂素处理的染色质纤维仅在600 mM - NaCl时发生破坏，而对照在500 mM - NaCl时就发生破坏。所有光反应样品的DNA在变性条件下铺展用于电子显微镜观察。它们显示，对于所有样品，单链气泡大小对应于200至400个碱基对。含有化学计量核心组蛋白的H1缺失染色质在pH 10和极低离子强度下进行光反应。在这些条件下，许多核小体似乎被解开，尽管程度不同。在电子显微镜下，这些样品纯化的DNA在变性条件下铺展时显示出广泛的交联。这些观察结果表明，可能会产生与完整核小体中不同的组蛋白 - DNA相互作用，这使得补骨脂素能够广泛地接触到DNA。
