Reactive oxygen species produced by ionizing radiation introduce a variety of lesions in DNA including strand breaks, abasic (AP) sites, and fragmented deoxyribose forms including 3-phosphoglycolate esters and 3'- phosphomonoesters. These modified 3'-termini block DNA synthesis if not identified by a class or direct acting enzymes called AP endonucleases that are capable of removing blocked 3'-termini, as well as initiating the repair of unmodified AP sites that arise spontaneously or as the product of DNA glycosylases. Since AP sites are potentially mutagenic, AP endonucleases are important enzymes to restore genetic integrity.
The aims outlined in this proposal are intended to broaden our knowledge or abasic DNA repair in the genetically well-established eukaryotic organism Drosophila melanogaster. Three AP endonucleases in Drosophila will be studied, namely AP3, AP endonuclease I, and AP endonuclease fl. The cDNA encoding AP3 has already been cloned. AP3 is bound to both nuclear matrices as well as ribosomes. Its human homologue is ribosomal phosphoprotein PO, which is elevated in patients suffering from the autoimmune disease lupus. PO is also elevated in certain DNA-repair deficient human tumor cell lines, thus drawing a link between certain autoimmune diseases and DNA repair. The spectra of DNA damage recognized by this AP endonuclease will be evaluated using a number of different modified DNA substrates including lesions produced by ionizing radiation. The other two AP endonucleases will be cloned using either an antibody that specifically cross-reacts with these proteins, or oligonucleotides generated to specific regions of the purified proteins. Once cloned, products will be tested for biochemical activity similar to tests used for defining the lesions identified by AP3. All three cDNA's will be used to identify the genomic organization and upstream regulatory regions associated with these enzymes and allow us to examine how these genes respond to various environmental stimuli. Genetically, we will establish the chromosomal location of these genes through in situ hybridization, and from the knowledge gained from these studies target our search for mutants deficient for each of the AP endonucleases in Drosophila. Mutants identified will be subjected to various environmental agents to determine which of these proteins dominates for a particular repair pathway. Likewise, the rescue of E.coli mutants deficient for each of the AP endonucleases will provide important information on the major role each of these AP endonucleases play in the repair of DNA damaged by agents generating reactive oxygen species along with alkylation damage.
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