The specific aims of this research are to the structure and function of endonuclease III of Escherichia coli and to identify endonuclease III - like enzymes in other organism. Three major approaches to study the structure and function of endonuclease III will be used. Firstly, site directed mutagenesis will be employed to alert a small number of specific amino acid residues suggested from previous studies to play a major role in enzyme activity and the mutant enzymes will be examined using biochemical techniques. Secondly, the binding of endonuclease III to damaged DNA will be studies using synthetic oligonucleotides containing a unique damaged base of AP site. Footprinting experiments and chemical protection experiments will give information about specific binding of enzyme to DNA, about specific contacts with bases and phosphate backbone and will allow an estimate of the binding constant of the enzyme to damaged DNA. Thirdly, endonuclease III will be crystallized and the three-dimensional structure of the protein will be determined by X-ray crystallography. If possible, the enzyme will be co-crystallized with one or more of the synthetic oligonucleotides mentioned above for X-ray analysis. Antibodies will be used as a means of identifying endonuclease III-like enzymes in E. coli and other organisms. The cloning, nucleotide sequencing of the genes for these enzymes and purification of these enzymes will yield useful information about the conservation of the Fe-S center and residues or domains that seem to be critical for enzymatic function.
