Active oxygen and free radicals arise constantly from both environmental sources, such as ionizing radiation, and normal physiology, and they cause damages to cellular macromolecules. Oxidative damage to DNA is both lethal and mutagenic in human cells. The mechanism and control of the repair of this damage in humans is largely unknown. The many 3'-terminal fragments that arise from oxidative attack require efficient excision to allow DNA repair synthesis. Such broken termini might lead to genetic rearrangement or the induction of emergency responses (e.g., poly(ADP-ribose) production) if left unrepaired. We plan to purify the human diesterase responsible for such 3'-terminal processing. HeLa cells have already been identified as a ready source of an abundant activity that is distinct from known enzymes. Possible regulation of this diesterase by exposure of the cells to DNA- damaging agents (e.g., X-rays) will be tested. The enzyme purification will employ a variety of standard chromatographic techniques, and we will also develop affinity methods. The purified enzyme will be characterized for its physical and kinetic properties, with special emphasis on its specificity for different damages in DNA. With the purified protein in hand, we will also work to clone the protein's complementary DNA and then its structural gene, using both immunological methods and information from the polypeptide sequence. The cloned DNA will be sequenced and examined for homology to known genes, and checked for cross-hybridization to the DNA of other eukaryotes. Transient inhibition of diesterase synthesis might also be achieved using constructs that generate antisense RNA. Ultimately we plan to generate human cell lines in which the normal diesterase structural gene has been inactivated by targeted recombination. These experiments will provide a detailed view of the biochemistry, genetics and cell biology of oxidized DNA repair. This information is essential to our understanding of normal genetic stability and the role of one type of oxidative and radiation damages in the origins of some human cancer.
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