Brooks W, Jackson V
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
J Biol Chem. 1994 Jul 8;269(27):18155-66.
The interaction of histones H2A and H2B with closed circular DNA maintained in either a positive or negatively coiled state has been studied. The methods involved separation of complexes by sucrose gradients and by agarose gel electrophoresis. Histones H2A and H2B have a 10-fold preference for DNA that contains performed negative coils as compared to DNA that is positively coiled or topologically unrestrained. It was also observed that histones H2A and H2B, when initially reconstituted onto positively coiled DNA, transfer very efficiently to negatively coiled DNA at 100 mM NaCl. Transfer is virtually complete within 5 s at 4 degrees C. By utilizing a cross-linking agent (disuccinimidyl suberate), it was found that histones H2A and H2B could be cross-linked into a tetrameric structure when on negatively coiled DNA but not on positively coiled DNA. It is concluded that protein-protein interactions facilitate the transfer. The importance of these protein-protein interactions is further demonstrated in experiments in which H3,H4 were included, and, in this instance, the strong interaction of H2A,H2B with H3,H4 negates the preference of H2A,H2B for negatively coiled DNA. These observations are discussed with regard to the mobility of histones during the transcription process during which transcription-induced stresses may be produced.
已经研究了组蛋白H2A和H2B与保持正超螺旋或负超螺旋状态的闭环DNA之间的相互作用。所采用的方法包括通过蔗糖梯度和琼脂糖凝胶电泳分离复合物。与正超螺旋或拓扑结构不受限制的DNA相比，组蛋白H2A和H2B对含有预形成负超螺旋的DNA具有10倍的偏好性。还观察到，当组蛋白H2A和H2B最初重组到正超螺旋DNA上时，在100 mM NaCl条件下能非常有效地转移到负超螺旋DNA上。在4℃下，转移在5秒内几乎完成。通过使用交联剂（辛二酸二琥珀酰亚胺酯）发现，当组蛋白H2A和H2B结合在负超螺旋DNA上时可以交联成四聚体结构，而结合在正超螺旋DNA上时则不能。得出的结论是，蛋白质 - 蛋白质相互作用促进了转移。在包含H3、H4的实验中进一步证明了这些蛋白质 - 蛋白质相互作用的重要性，在这种情况下，H2A、H2B与H3、H4之间的强烈相互作用消除了H2A、H2B对负超螺旋DNA的偏好。针对转录过程中组蛋白的移动性对这些观察结果进行了讨论，在转录过程中可能会产生转录诱导的应力。
