The genetic material, DNA, is inherently unstable and also subject to attack by both metabolic by-products and extracellular agents. Key environmental and endogenous DNA-damaging mutagens include reactive oxygen species and simple alkylating agents. These cause the loss of nucleotides or bases both directly and indirectly to leave various types of apurinic/apyrimidinic (AP) sites. These AP sites represent a loss of genetic information and can be mutagenic and perhaps carcinogenic. The enzymes that initiate the repair of AP sites and related damages, the AP endonucleases, are ubiquitous in biology. Their biological functions have been established only in microorganisms (E. coli and yeast), but they are likely to be front-line defense enzymes in human cells as well. Our goal is the definition of the biological role of the major human AP endonuclease, encoded by the APE gene whose cDNA we recently cloned, and the molecular cloning of the gene encoding a new human AP endonuclease we recently discovered. We will isolate genomic clones of APE and use these probes to determine the gene's physical position in the human genome. We will examine the expression of the APE gene in cultured cells and develop reporter vectors with the CAT gene to monitor APE expression through the cell cycle and after treatment of cells with DNA- damaging and other stressful agents. In order to probe the cellular function of the enzyme, we will engineer Ape-deficient and Ape- overexpressing cell lines using sense and antisense expression vectors. These lines will be examined for their sensitivity to the cell-killing effects of oxidative and other agents. These sensitivities will be correlated with DNA damages in chromosomal DNA from the treated cells. We will determine whether the APE AP endonuclease contributes to genetic stability by examining spontaneous and mutagen-induced mutation in these lines. We will also develop molecular probes for the new AP endonuclease gene, the cloning of which will allow us to apply similar tests of that enzyme's function in the maintenance of genetic integrity.
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