title: Measuring Equilibrium Binding Constants for the WT1-DNA Interaction Using a Filter Binding Assay.
abstract: Equilibrium binding of WT1 to specific sites in DNA and potentially RNA molecules is central in mediating the regulatory roles of this protein. In order to understand the functional effects of mutations in the nucleic acid-binding domain of WT1 proteins and/or mutations in the DNA- or RNA-binding sites, it is necessary to measure the equilibrium constant for formation of the protein-nucleic acid complex. This chapter describes the use of a filter binding assay to make accurate measurements of the binding of the WT1 zinc finger domain to the consensus WT1-binding site in DNA. The method described is readily adapted to the measurement of the effects of mutations in either the WT1 zinc finger domain or the putative binding sites within a promoter element or cellular RNA.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Road, Victoria, BC, Canada, V8P 5C2. pjr@uvic.ca.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Romaniuk
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D025721:WT1 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: D025721:WT1 Proteins; C583751:WT1 protein, human; D012313:RNA; D004247:DNA
keywords: DNA-protein; Equilibrium binding; Filter binding assay; Protein-nucleic acid interactions; RNA-protein; WT1; Zinc finger protein