This project aims to unravel the three-dimensional structures as well as the mode of operation of two DNA-binding proteins: diphtheria toxin repressor from Corynebacterium diphtheria, the causative agent of diphtheria, and human topoisomerase I. The key question to be addressed regarding diphtheria toxin repressor is its regulation of sequence-specific DNA-recognition by transition metals, in particular Fe2+. No three-dimensional structures of metal-regulated repressors are known yet, hence the goal is to elucidate crystal structures of the repressor in complex with duplex DNA and a series of transition metal ions. Fe-dependent repressors play an important role in many pathogenic bacteria, such as Shigella dysentheriae and Vibrio cholerae, where they control the role in many pathogenic bacteria, such as Shigella dysentheriae and Vibrio cholerae, where they control the expression of several virulence factors. Consequently, the three dimensional structure of diphtheria toxin repressor will be a starting point for the design of small molecules which will enhance the affinity of the repressor for the operator, Such molecules not only prevent the production of toxin but also impede the growth of bacteria by suppressing the production of other proteins which are essential for pathogen. Human topoisomerase I is important for maintaining the proper higher order topology of DNA during transcription, replication and recombination. The specific goals are to elucidate the three-dimensional crystal structures of the enzyme, wild type and mutants, with and without DNA bound, in the presence of several inhibitors. The results provide a firm basis for unraveling the complicated catalytic mechanism of the enzyme and for understanding the mode of action of a wide variety of inhibitors. Several of these topoisomerase I poisons ar e of the greatest interest as they have very promising properties for the treatment of a wide variety of cancers. In addition, the crystal structure of human topoisomerase I will form an excellent basis for the design of new inhibitors which ar potential new cancer drugs.
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