Structural and kinetic study of the self-assembly of nucleosome core particles.
Structural and kinetic study of the self-assembly of nucleosome core particles.
复制标题
核小体核心颗粒自组装的结构和动力学研究。
DOI：
10.1016/0022-2836(82)90140-1
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发表时间：
1982
影响因子：
5.6
通讯作者：
Cantor,CR
中科院分区：
文献类型：
--
作者：
Daban,JR;Cantor,CR
当分离的鸡红细胞核心组蛋白（或在2.0m-NaCl中解离的核心颗粒）在pH 7.0下用荧光试剂n -（l-芘）马来酰亚胺标记时，只有组蛋白H3发生反应，可能是在半胱氨酸110处。pyrenee标记的岩心颗粒从2.0m-NaCl跳盐到更低的浓度。当最终NaCl浓度大于0.6m时，只观察到芘单体发射，而在0.6 ~ 0.2m之间，单体强度降低，而芘准分子强度增加，说明两个芘环在紧凑的核小体结构中可以相互接触。准分子发射提供了一种直接和简单的方法来监测核小体的组装。DNA酶I酶切和光谱研究表明，通过2.0 ~ 0.2m-NaCl的跳盐或直接在0.2m-NaCl中混合DNA和组蛋白，可以重构核心颗粒，但前者效率更高。通过测量准分子发射量的增加，研究了从2.0 m- nacl跳盐到0.2m-NaCl或在0.2m-NaCl中直接混合的核粒子自组装动力学。得到的动力学数据可拟合为双指数速率方程（速率常数为~10−1s−1和~7 × 10−3s−1）。在2.0 ~ 0.2m-NaCl盐跳和0.6 ~ 0.2m-NaCl盐跳的动力学参数基本相同，说明在0.6m- nacl盐跳以上，组蛋白与DNA的结合速度非常快，整个反应中观察到的两种动力学成分都与核心颗粒的折叠有关。动力学参数与DNA和组蛋白浓度无关，排除了涉及二级反应的机制。两步顺序机制与折叠动力学一致，但对展开-再折叠反应的进一步研究表明，必须考虑更复杂的机制。涉及两个中间体的线性或循环机制与现有的动力学数据一致。这些中间体可能与在新复制的染色质中观察到的核小体结构的改变有关，或者它们可能参与染色质转录。
When isolated chicken erythrocyte core histones (or core particles dissociated in 2.0m-NaCl) are labeled with the fluorescent reagentN-(l-pyrene)maleimide at pH 7.0, only histone H3 reacts, presumably at cysteine 110. Pyrene-labeled core particles were salt-jumped from 2.0m-NaCl to lower concentrations. When the final NaCl concentration is higher than 0.6m, only pyrene monomer emission is observed, whereas between 0.6 and 0.2m, the monomer intensity decreases, while pyrene excimer intensity increases, indicating that the two pyrene rings can contact each other in the compact nucleosome structure. The excimer emission provides a direct and simple way to monitor nucleosome assembly. Digestion with DNAase I and spectroscopic studies show that core particles can be reconstituted either by salt-jump of core particles from 2.0 to 0.2m-NaCl or by direct mixing of DNA and histones in 0.2m-NaCl, but the former procedure is more efficient.The kinetics of core particle self-assembly by salt-jump from 2.0 to 0.2m-NaCl or direct mixing in 0.2m-NaCl were followed by measuring the increase in excimer emission. The kinetic data obtained can be fitted to a two-exponential rate equation (rate constants: ~10−1s−1and ~7 × 10−3s−1). The kinetic parameters are essentially the same for a 2.0 to 0.2m-NaCl salt-jump and a 0.6 to 0.2m-NaCl salt-jump, indicating that binding of histones to DNA, which takes place above 0.6m-NaCl, is very fast and that the two kinetic components observed for the overall reaction are related to the folding of the core particle. The kinetic parameters are independent of DNA and histone concentrations, excluding mechanisms involving second-order reactions. A two-step sequential mechanism is consistent with the folding kinetics but further investigations on unfolding-refolding reactions indicate that more complex mechanisms must be considered. Linear or cyclic mechanisms involving two intermediates are consistent with the available kinetic data. These intermediates could be related with the altered nucleosome structures observed in newly replicated chromatin or they may be involved in chromatin transcription.