DIFFRACTION ANALYSES OF SS DNA COMPLEXES WITH PROTEINS

点击翻译按钮获取中文摘要

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.