Structural Studies of Histone - DNA Interactions

本研究的目标是：(1)单个组蛋白H1变异的表达，(2)定量它们与定义序列DNA的相互作用，(3)利用CD光谱确定组蛋白结合对DNA构象的影响，以及(4)确定各种H1结构域结构在与DNA相互作用中的作用。这项工作的结果将有助于澄清相对非特异性DNA结合蛋白与DNA相互作用的性质。这些研究将为组蛋白：DNA相互作用中结构-功能相关性的本质提供初步指示。真核生物的复杂性决定了对大型DNA结构的需求，这些结构只能通过与蛋白质（如组蛋白）的相互作用来容纳在有限体积的活细胞中，组蛋白将DNA包裹成紧密排列的结构，但仍然允许细胞的生物合成DNA的机器。提出的研究调查了自然发生的组蛋白H1变异之间的相互作用。组蛋白极难纯化。这项研究涉及到分子生物学方法产生的纯组蛋白的新用途。将检查组蛋白与多种DNA结构的亲和力，并在一定程度上确定复合物中组蛋白和DNA的结构。比较各种组蛋白变体与DNA结构的亲和力，将建立组蛋白结构与其对DNA亲和力之间的关系。组蛋白与DNA结构的相互作用是一个重要的研究课题，因为DNA组蛋白复合物在控制活细胞的功能、发育和成熟方面具有潜在的重要性。

The goals of this study are (1) expression of individual histone H1 variants, (2) quantitation of their interactions with DNA of defined sequence, (3) determination of the effects of histone binding on the conformation of DNA utilizing CD spectroscopy, and (4) determination of the roles of various H1 domain structures in the interaction with DNA. The results of this work will help clarify the nature of the interactions of relatively non-specific DNA-binding proteins with DNA. These studies will provide a preliminary indication of the nature of structure-function correlations in histone:DNA interactions. %%% The complexity of eucariotic organisms dictates a requirement for large DNA structures which can only be accomodated in the limited volume of the living cell by means of interactions with proteins such as the histones which wrap the DNA into tightly packed structures which nonetheless permit access of the cell's biosynthetic machinery to the DNA. The studies proposed investigate the interaction of naturally occurring variants of histone H1. The histones are extremely difficult to purify. This study involves the novel use of pure histones produced by molecular biological methods. The affinity of the histones with a variety of DNA structures will be examined and to some extent the structures of the histones and DNA in the complexes will be determined. Comparison of the affinity of the various histone variants with DNA structures will establish the relationship between the structures of histones and their affinity for DNA. The interaction of histones with DNA structures is an important subject for study because of the potential importance of DNA:histone complexes in controlling the function, development, and maturation of living cells.