Human cells contain singular DNA repair pathways to excise critical lesions from DNA as a mechanism to prevent the initiation of neoplasia. Alterations in these pathways may result in the failure to remove miscoding lesions thus facilitating tumor development. Alternatively, as many cancer therapeutic agents are themselves DNA damaging agents, tumor cells may utilize the identical pathways to mitigate the efficacy of cancer therapy. Accordingly, this research proposal is designed to probe the basic mechanisms through which human cells recognize and remove critical DNA adducts. Investigation of the base excision repair enzyme uracil DNA glycosylase will be used as a model system. These studies will use the highly purified normal human enzyme; natural mutant(s) of the enzyme isolated from individuals with Bloom's syndrome, a human disease characterized by a high cancer rate; and a panel of anti-human uracil DNA glycosylase monoclonal antibodies. The objectives of this proposal are: 1) To examine the immunology and sequence homology of human uracil DNA glycosylases. Enzymes structure will be examined by peptide mapping and identification of the glycosylase monoclonal antibody binding sites. The specific amino acid alteration(s) in the Bloom's syndrome enzyme will be determined and related directly to alterations in the nucleotide sequence of the human uracil DNA glycosylase gene. 2) To examine the basic mechanisms through which this miscoding lesion is removed from DNA. These investigations will include kinetic analysis of the catalytic reaction as well as the mechanism through which 5- flyorouracil, a cancer chemotherapeutic agent, inhibits glycosylase activity. These studies will use both the normal and mutant human enzymes. These experiments will define the relationship between alterations in protein structure and the ability of human DNA repair enzymes to prevent the initiation of neoplasia by the removal of critical DNA lesions.
Showing the most recent 10 out of 23 publications
