To understand the molecular basis for the repair of cancer-causing mutations from damaged DNA and the molecular mechanisms for the metalloenzymes that perform DNA repair, we have successfully initiated integrated structural and biochemical studies for three major excision- repair metalloenzymes: endonuclease III, exonuclease III, and endonuclease IV from E. coli. Genes for these three metalloenzymes, which represent the two major classes of base-excision repair endonucleases in eucaryotic and procaryotic cells, have been cloned and overexpressed in E. coli, allowing the proposed site-directed mutagenesis experiments and large scale purification for biochemical and crystallographic experiments. We have crystallized endonuclease III, determined the space group of the crystals, identified the position of the Fe4S4 cluster by solution of an anomalous Patterson map, and collected native data to 1.7 lambda resolution. Exonuclease III has been overexpressed, purified, and crystallized. Endonuclease IV has been overexpressed and partially purified. These accomplishments will allow the proposed comprehensive structure/function studies of these metalloenzymes, using the techniques of molecular biology, biochemistry, x-ray crystallography, and computational analysis. We propose to determine the atomic structure for endonuclease III, to prepare inhibitors and substrate analogs (such as thymine glycol), and to determine structures for their complexes with the enzyme. We will design, construct, and produce site-directed mutants of endonuclease III, and characterize them biochemically and crystallographically. Using the results of these studies, we will characterize the mechanisms for the glycosylase and apurinic/apyrimidinic (AP) endonuclease activities of endonuclease III. Enzymatic, crystallographic, and computational studies will be combined to understand the chemical and structural basis for the recognition and discrimination of damaged DNA by endonuclease III. Parallel studies on exonuclease III and endonuclease IV will be designed to complement the data from endonuclease III. The proposed research on these metalloenzymes for DNA repair is intended to probe structural determinants for their biological activity, enzyme mechanisms, and their recognition and discrimination of damaged and undamaged DNA. This knowledge should contribute specifically to understanding the role of DNA repair enzymes in the prevention of cancer-causing mutagenesis from oxidative damage and other causes, and more generally to defining the atomic basis for protein- DNA recognition processes fundamental to cell growth and differentiation. Due to the widespread use of radiation treatment for cancer, an important potential medical application of the proposed research would be the design of endonuclease inhibitors to increase radiation sensitivity of tumors, and thus decrease radiation doses and damage to normal tissue.
Showing the most recent 10 out of 63 publications
