A combination of 2D NMR techniques will be used to study the solution state structure of three homologous type II DNA-binding proteins and their interaction with DNA. One member of this family, HU, has been crystallized and the structure of this protein provides a basis for analyzing the DNA binding properties of other DBP-II proteins. The class of proteins appear to bind to DNA in a manner that is quite different from the other regulatory proteins that have been studied in that """"""""arms,"""""""" approximately 30 residues in length, appear to interact with the DNA. The TF1 protein, a viral homologue of the bacterial DBP-II is of particular interest because it preferentially interacts with phage SPO1 DNA containing hydroxymethyluracil, and binds to specific sequences of SPO1. Biochemical and fluorescence studies show that there are important differences between the interaction of TF1 and SPO1 DNA and calf thymus DNA and there is strong evidence that the terminal 9 amino acid residues are largely responsible for these differences. Integration host factor (IHF), a second member of this class is of interest because it participates in a number of different processes, including recombination and the regulation of some E. coli genes. HU is of interest because the crystal structure of this protein is available for comparison with solution state studies. Finally, studies of the structure of the small (37 amino acids) J protein of bacteriophage phiX174 will be carried out. This protein is important in the packaging of the viral DNA to produce infectious phage and is not in the same class as the other DNA binding proteins. Studies of the structure and DNA binding properties of the type II DNA-binding proteins are important because they are ubiquitous in prokayrotes and their mode of binding to DNA is quite different from other regulatory proteins that have been studied in more detail. The studies proposed here will contribute to our understanding of this alternative mode of DNA binding and provide insight into the factors responsible for sequence specific binding to DNA. Two dimensional NMR techniques are the primary tools to be used in the structural studies and these will be used in conjunction with fluorescence polarization anisotropy measurements to study the DNA interaction. As part of our TF1 studies, the properties of hydroxymethyluracil containing DNA duplexes will be compared with thymine containing DNA of the same sequence.
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