Aragay A M, Diaz P, Daban J R
Departament de Bioquímica i Biologia Molecular Facultat de Ciències, Universitat Autònoma de Barcelona, Spain.
J Mol Biol. 1988 Nov 5;204(1):141-54. doi: 10.1016/0022-2836(88)90605-5.
In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 M). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 M-NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.
在非变性低离子强度凝胶中，用H2A、H2B对核心DNA进行滴定会产生五条清晰可辨的条带。定量光密度测定和交联实验表明，这些条带是由于H2A、H2B二聚体与核心DNA的相继结合。DNA-(H3、H4)样品仅得到两条条带。其中较慢的条带较宽，可能分别对应于含有一个和两个H3、H4四聚体的两种复合物。在离子强度较高的凝胶中，DNA-(H2A、H2B)样品产生一条模糊不清的条带，这表明含有H2A、H2B的复合物的寿命相对较短。然而，在这些凝胶中观察到的游离DNA条带强度较低，这表明大多数DNA与H2A、H2B结合。与此一致的是，我们使用不同技术（沉降、交联、胰蛋白酶和核酸酶消化以及热变性）获得的结果表明，无论有无NaCl（0.1至0.2M），H2A、H2B与核心DNA的结合都发生在游离溶液中。DNA-(H2A、H2B)复合物的低迁移率，以及沉降和DNase I消化结果表明，这些复合物中的DNA没有折叠成核心颗粒中发现的紧密结构。此外，非变性凝胶已被用于研究0.2M-NaCl中DNA-(H2A、H2B)和DNA-(H3、H4)复合物的动态特性。我们的结果表明：(1) H2A、H2B和H3、H4可以分别与DNA-(H3、H4)和DNA-(H2A、H2B)结合，形成含有四种核心组蛋白的复合物；(2) DNA-(H2A、H2B)和DNA-(H3、H4)能够将组蛋白转移到游离的核心DNA上；(3) DNA-(H2A、H2B)和DNA-(H3、H4)之间发生组蛋白对的交换，产生与正常核小体核心颗粒具有相同组蛋白组成的复合物；(4) 尽管两种组蛋白对都可以交换，但H2A、H2B组蛋白比H3、H4组蛋白更倾向于从一个不完全核心颗粒迁移到另一个。这些反应中产生的复合物具有与含有四种核心组蛋白的重组核心颗粒相同的紧密结构。我们的动力学结果与一种反应机制一致，即组蛋白的转移涉及反应复合物之间的直接接触。讨论了这些自发反应在核小体组装机制中的可能参与情况。
