Characterization of Protein-DNA Complexes

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DESCRIPTION: (provided by applicant) Chromosomal proteins bind to DNA in a nonsequence-specific fashion, but the mechanism by which these proteins recognize DNA is poorly understood. The DNA complexes of chromosomal proteins are involved in mediating cellular processes. Therefore, a better understanding of the binding interactions of these complexes better enable clinicians to address disorders whose origin may be related to these interactions. It has been suggested that the reasons for the non-specificity lies in the interaction of two principal loci of the protein with DNA. Proposed here is a study to test this hypothesis through three specific aims. The first aim is to design and produce a sequence-specific protein from a nonsequence-specific protein scaffold. The second is to test the specificity of the designed protein to DNA. Thirdly, we propose to examine the interactions that are important for specificity by determining the three-dimensional structure of the designed protein in complex with DNA. The DNA scaffold for HMG-D, a nonsequence-specific protein, can be genetically engineered so that sequence-neutral residues will be replaced by residues which recognize specific-sequences, resulting in the putative protein, SPEC. The binding site will be evaluated by a electrophoretic mobility shift assay (EMSA) using oligomers of random sequence. Competitive band shift experiments employing a long chain probe DNA and competitor oligomers will be used to ascertain the binding site sequence and length. DNase I footprinting assays will be used to further evaluate the sequence specificity by SPEC. The three-dimensional structure of the complex will be determined by first co-crystallizing SPEC and duplex DNA, followed by collection and analysis of X-ray diffraction data.