A combination of 2D and 3D homo- and heteronuclear NMR techniques will be used to study the solution state structure of a type II DNA-binding protein (DBPII) and its interaction with DNA. One member of this family has been crystallized and the structure of this protein provides a basis for analyzing the structures and DNA-binding properties of other DBPII proteins. This family of proteins appears to bind to DNA in a manner that is quite different from the other regulatory proteins that have been studied in that two beta-ribbon """"""""arms,"""""""" approximately 30 residues in length, are hypothesized to interact with the DNA. The TF1 protein, a viral homolog of the bacterial DBPII is of particular interest because it preferentially interacts with DNA containing hydroxymethyluracil (hmU), and binds to specific sequences of phage SPO1 DNA. 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 nine amino acid residues are largely responsible for these differences. A large protein on the NMR scale, the quality of the spectra of TF1 has been greatly improved, both in terms of resolution and quantitative interpretation, through the use of selective deuteration and uniform isotopic labeling with 2h and l5n, as well as utilizing multidimensional techniques. This combined strategy has made it possible to make considerable progress towards the complete sequence-specific resonance assignment of TF1, the first step in the elucidation of its solution structure. 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 the alternative mode of DNA binding and provide insight into the factors responsible for sequence specific binding to DNA. Multidimensional NMR techniques are the primary tools to be used in the structural studies of the molecules and their interactions. 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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