PREFERENTIAL BINDING OF HISTONES H3 AND H4 TO HIGHLY POSITIVELY COILED DNA
PREFERENTIAL BINDING OF HISTONES H3 AND H4 TO HIGHLY POSITIVELY COILED DNA
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DOI：
10.1021/bi00033a036
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发表时间：
1995-08-22
期刊：
影响因子：
2.9
通讯作者：
JACKSON, V
中科院分区：
文献类型：
--
作者：
JACKSON, V
研究了组蛋白H3和H4与高度正螺旋DNA的相互作用。为了进行这项研究，有必要开发一种用于制备大量高度正螺旋DNA的方案。通过在含有PBR 322的细胞中最大化转录诱导的应激，然后应用抑制促旋酶的新生霉素进行短期治疗来获得这种制剂。在CsCl-EtBr梯度上的分级得到拓扑状态范围为+0.15至-0.043超螺旋密度的PBR 322质粒。在负螺旋DNA和正螺旋DNA的竞争实验中，当超螺旋密度大于+0.10时，组蛋白H3、H4优先结合正螺旋DNA。这种偏好的基础上显示的蔗糖梯度，选择性聚集H3，H4结合，交联实验中，其特征在于CsCl密度梯度的组蛋白-DNA含量的沉降速率。在1.1 M NaCl中的DNA螺距的分析表明，这种优先结合可能是由于螺距减少了约0.06 bp/tum。与这一观察结果一致的是组蛋白H3、H4在拓扑异构酶I存在下瞬时保持这种改变的音高的能力。对这种偏好的一个可能的解释是基于已知的观察，即组蛋白在核小体中时会过度缠绕DNA螺旋。这些数据提供了一个解释，在体内观察组蛋白H3，H4在转录过程中很少被取代。这些意见进行了讨论，通过核小体转录的机制。
The interaction of histones H3 and H4 with highly positively coiled DNA has been studied. To carry out this study, it was necessary to develop a protocol for the preparation of large quantities of highly positively coiled DNA. Such preparations were obtained by maximizing transcription-induced stress in cells containing PBR322 and then applying a short-term treatment with novobiocin, which inhibits gyrase. Fractionation on CsCl-EtBr gradients gave PBR322 plasmids with topological states ranging from +0.15 to -0.043 superhelical density. In competition experiments between negatively and positively coiled DNA, histones H3,H4 preferentially bound the positively coiled DNA when the superhelical density was greater than +0.10. This preference was shown on the basis of sedimentation rate on sucrose gradients, selective aggregation by H3,H4 binding, and cross-linking experiments in which the histone-DNA content was characterized on CsCl density gradients. An analysis of the DNA helical pitch in 1.1 M NaCl, a condition in which moderately positively coiled DNA preferentially binds H3,H4, indicated that the preferential binding may be due to a decrease in helical pitch that approximates 0.06 bp/tum. Consistent with this observation is the ability of histones H3,H4 to transiently hold this altered pitch in the presence of topoisomerase I. A possible explanation for this preference is based on the known observation that histones overwind the DNA helix when in a nucleosome. These data provide an explanation for the in vivo observation that histones H3,H4 are rarely displaced during the transcription process. These observations are discussed with regard to mechanisms for transcription through nucleosomes.