DIFFRACTION ANALYSES OF SS DNA COMPLEXES WITH PROTEINS

两个单链DNA（ssDNA）结合的进一步研究 蛋白质和这些蛋白质与ssDNA的复合物， 提出了 在第一种情况下，基因5 DNA的结构 来自噬菌体FD的解旋蛋白质将被精制到1.5 目前使用X射线衍射数据的埃分辨率 手 这将允许更精确地定义DNA结合 界面和相关氨基酸侧链以及 水化层的划分， 核酸 基因5的一种新的高度水合晶体形式 蛋白质将通过分子置换技术解决， 用于形成基因5-脱氧寡核苷酸 配合物 这些将允许直接可视化基因5 蛋白质-DNA相互作用 我们将继续努力， 基因5蛋白与脱氧寡聚体的复合物， 适合X射线衍射分析。 第二个要研究的ssDNA结合蛋白是牛RNA酶 A和B。 我们已经发现这种DNA解旋蛋白可以 与大量不同的脱氧寡聚体复合 包括d（pA）4、d（pT）4、d（pA）6，并且这些络合物可以 结晶为类质同晶系列。 我们已经解决了 其中几个结构，并表明，在所有情况下， 不对称单位由一个蛋白质分子加上三个 五个脱氧寡聚体。 脱氧寡聚体本身形成 各种复杂的网络连接，在某些情况下螺旋 晶体中的线。 我们建议研究一系列 蛋白质-DNA晶体复合物，以评估结构 ssDNA的性质，它与蛋白质的相互作用及其自身的性质， 交互. 我们还打算使用傅立叶差分 技术来研究一个广泛的 致癌物、诱变剂、锥虫甙、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ssDNA复合物。 通过检查广泛的 我们打算描述化学物质， 和结构因素负责的特异性和有效性 与蛋白质-核酸复合物相互作用的药物。 将牛RNase基因克隆到合适的表达载体中， 载体和定点诱变用于引入特异性 修改. 改变后的蛋白质分子 通过X射线衍射显示为与DNA的结晶复合物 寡聚体，以进一步描绘个体的机械作用， DNA结合中的氨基酸。 此外，我们还将尝试制备其他ssDNA的晶体 适合于X射线结构分析形式的结合蛋白。 这些将包括来自E.杆菌 RecA蛋白，lac阻遏蛋白，T4噬菌体基因32 蛋白质和E.大肠杆菌拓扑异构酶1。 这些蛋白质的晶体 这将使我们能够将我们的分析扩展到其他系统， 我们对蛋白质相互作用原理的理解 ssDNA。

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.