DIFFRACTION ANALYSES OF SS DNA COMPLEXES WITH PROTEINS

双单链DNA(单链DNA)结合的进一步研究 蛋白质以及这些蛋白质与单链DNA的复合体 建议。在第一种情况下，基因5 DNA的结构 Fd噬菌体解离蛋白将提纯至1.5 使用当前在#年的X射线衍射数据进行Angstrom分辨率 手。这将允许更精确地定义DNA结合 界面和相关的氨基酸侧链以及 与之相关的水化层的描绘 核酸。基因5的一种新的高度水合的晶体形式 蛋白质将通过分子替代技术和 用于基因5-脱氧寡核苷酸的形成 复合体。这些将允许直接可视化基因5 蛋白质与DNA的相互作用。将继续努力实现共结晶 基因5蛋白与脱氧低聚物的复合体 适用于X射线衍射分析。 第二个要研究的单链DNA结合蛋白是牛核糖核酸酶 A和B。我们已经发现，这种DNA解离蛋白可以 与大量不同的脱氧齐聚物形成络合物 包括d(PA)4、d(PT)4、d(PA)6，并且这些络合物可以 结晶成同构的系列。我们已经解决了 其中几个的结构，并表明在所有情况下 不对称单位由一个蛋白质分子加三个蛋白质分子组成。 到五个脱氧寡聚体。脱氧低聚物本身形成 各种复杂的相互连接的网络，在某些情况下是螺旋形的 晶体中的一股股。我们建议对这些问题进行一系列研究 蛋白质-DNA晶体复合体的结构评价 单链DNA的性质及其与蛋白质的相互作用和自身 互动。我们还打算使用差分傅里叶 技术来研究广泛的 一系列致癌物、诱变剂、胰黄素、抗生素、金属 离子和其他生理上重要的配体与蛋白质- 这些晶体中的单链DNA复合体。通过检查广泛的范围 药理药剂，我们打算描述化学物质 以及影响其特异性和有效性的结构性因素。 与蛋白质-核酸复合体相互作用的药物。 牛核糖核酸酶基因将被克隆到合适的表达中 利用载体和定点突变技术导入特异体 修改。然后，改变的蛋白质分子将被 X-射线衍射法显示为与DNA的晶体络合物 低聚物进一步描述个体的机械作用 DNA结合中的氨基酸。 此外，我们还将尝试制备其他单链DNA的晶体 以适合X射线结构分析的形式结合蛋白质。 这些将包括来自大肠杆菌的单链DNA结合蛋白， RecA蛋白、Lac阻遏蛋白、T4噬菌体基因32 蛋白质和大肠杆菌拓扑异构酶1.这些蛋白质的晶体 将允许我们将我们的分析扩展到其他系统，并扩展 我们对蛋白质相互作用原理的理解 用单链DNA。

Further investigation of two single strand DNA (ssDNA) binding proteins and complexes of these proteins with ssDNA are proposed. In the first case, the structure of the gene 5 DNA unwinding protein from bacteriophage fd will be refined to 1.5 Angstrom resolution using X-ray diffraction data currently in hand. This will allow more precise definition of the DNA binding interface and relevant amino acid side chains as well as delineation of the hydration layers that mediate association with nucleic acid. A new, highly hydrated crystal form of the Gene 5 protein will be solved by molecular replacement techniques and utilized for the formation of Gene 5-deoxyoligonucleotide complexes. These will allow direct visualization of the Gene 5 protein-DNA interactions. Efforts will continue to cocrystallize complexes of the Gene 5 protein with deoxyoligomers that are suitable for X-ray diffraction analysis. The second ssDNA binding protein to be studied is bovine RNAse A and B. We have found that this DNA unwinding protein can be complexed with a large number of different deoxyoligomers including d(pA)4, d(pT)4, d(pA)6 and that these complexes can be crystallized as an isomorphous series. We have solved the structures of several of these and shown that in all cases the asymmetric unit is comprised of one protein molecule plus three to five deoxyoligomers. The deoxyoligomers themselves form varied complicated networks of linked and in some cases helical strands in the crystals. We propose to study a series of these protein-DNA crystalline complexes to evaluate the structural properties of ssDNA, its interaction with protein and its self- interactions. We further intend to use the difference Fourier technique to study the binding and interaction of an extensive array of carcinogens, mutagens, trypanosides, antibiotics, metal ions and other physiologically important ligands with the protein- ssDNA complexes in these crystals. By examining a broad range of pharmacological agents, we intend to delineate the chemical and structural factors responsible for the specificity and efficacy of drugs which interact with protein-nucleic acid complexes. The bovine RNase gene will be cloned into a suitable expression vector and site directed mutagenesis utilized to introduce specific modifications. The altered protein molecules will then be visualized by X-ray diffraction as crystalline complexes with DNA oligomers to further delineate the mechanistic role of individual amino acids in DNA binding. We will, in addition, attempt to prepare crystals of other ssDNA binding proteins in forms suitable for X-ray structural analysis. These will include the ssDNA binding protein from E. coli, the RecA protein, the lac repressor protein, the T4 phage Gene 32 protein and E. coli topoisomerase 1. Crystals of these proteins will permit us to extend our analyses to other systems and expand our understanding of the principles by which proteins interact with ssDNA.