Protein-nucleic acid interactions of Wilms' Tumor and TFIIIA zinc finger proteins




Hamilton, Tatyana

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This Ph.D. work represents the study of nucleic acid interactions of two zinc finger proteins: mammalian Wilms' tumor suppressor (WT1) and Xenopus transcription factor IIIA proteins (TFIIIA). WT1 is a putative transcriptional regulatory protein which is inactivated in a subtype of Wilm s' tumours. Using selection and amplification binding assay (SAAB) we determined that the highest affinity binding sites for WT1[-KTS] consist of a 12 base pair sequence GCG-TGG-GCG-(T/G)(G/A/T)(T/G). These sequences have a four-fold higher affinity for the protein than the nonselected sequence GCG-TGG -GCG-CCC , as measured by a quantitative nitrocellulose filter binding assay. The effects of Denys-Drash syndrome (DOS) point mutations on the DNA binding activity of WT1 were determined. SAAB assay revealed that none of the DDS mutant proteins give rise to a new sequence specificity. One mutation, R394W abolishes specific binding of the protein. The remaining mutations result in reduced DNA- binding activity, ranging from 1.4 to 14-fold, which suggests that even small changes in DNA-binding activity may precipitate the clinical phenotype of Denys-Drash syndrome. Comparative analysis of the DNA binding characteristics of Wilms' tumour and Early growth response proteins was conducted . The stoichiometry of the DNA-protein complexes, their stability to dissociation, and the effects of pH, temperature and salt concentration on the equilibrium binding of these proteins to their cognate DNA sequences have been determined. Under the conditions of 0.1 M salt, p H 7.5, and 22 * C WT1-ZF has an apparent dissociation constant (Kd) of 1.14± 0.2 X 10⁻⁰⁹ M, and EGR-1 protein has a Kd of 3.55 ± 0.4 x 10⁻⁰⁹ M. In addition, we tested relative contribution of each base pair in the consensus binding site to the high affinity binding by point mutational analysis, and identified important differences that exist in the binding modes of the two proteins. Transcription factor IIIA controls the expression of the 5S ribosomal RN A genes during development of Xenopus laevis., and specifically interacts with both 5S DNA and 5S rRNA molecules. The present study assesses contributions of the central zinc fingers four through seven to specific DNA and RNA binding activities of the protein. The results demonstrate that each zinc finger in the zf 4-7 region contributes to both the high affinity DNA and RNA interactions: the largest effect on TFlllA-DNA binding (10-fold) wa s produced when zinc finger 5 of TFIIIA was replaced with the donor sequences of either p43 or WT1. However, while all the zinc fingers 4-7 contribute to the high affinity 5S rRNA binding, substitution of an α- helical portion of zinc finger 6 with the equivalent sequences from WT1 abolished RNA-binding activity of TFIIIA, suggesting that zIinc finger 6 p lay s a particularly im portant role in binding to RNA.



Nucleic acids