DNA is inherently unstable, but also attacked by metabolites and environmental mutagens such as oxygen radicals and alkylating agents, which form various types of abasic (AP) sites. AP sites can be mutagenic and so compromise genetic stability. Repair of AP sites is initiated ubiquitous """"""""class II"""""""" AP endonucleases. Their biological functions have been tested in microorganisms, but they are likely front-line defense enzymes in humans, too. He will define the biological role of the main mammalian AP endonuclease, encoded by the APE gene, approaching this goal in several complementary ways. He will establish Ape-deficient cell lines from mouse embryonic tissues arising from mating between APE disruption (""""""""knockout"""""""") heterozygotes; homozygous knockout embryos do not develop beyond day 5, but cells derived from them can be cultured in vitro. The cell lines will be tested for sensitivity to mutagens associated with AP site production (e.g., alkylating agents), as well as other agents. Dr. Demple will measure spontaneous mutation frequencies for these lines to gain insight into the role of Ape in correcting endogenous DNA damage. He will test whether the phenotypes are reversed by introduction of recombinant wild-type APE, or mutant derivatives encoding proteins defective in repair or """"""""Refl"""""""" activity that affects transcription factor binding in vitro. He will also explore interactions of Ape with other repair and non-repair proteins using yeast two-hybrid and phage display methods. He have already demonstrated that Ape interacts with DNA polymerase B, which catalyzes the next two steps in base excision repair. He will explore the significance of this interaction by identifying regions of the two proteins needed for this interaction. Dr. Demple will isolate Ape derivatives unable to interact with the polymerase and assess their ability to support normal repair in vivo. Thus, this is a proposal to study the role of the mammalian AP endonuclease (the product of the Ape gene) in DNA repair and in restoration of the DNA binding activity oxidized c-jun or c-fos (the ref1 activity of Ape). The applicant proposes to generate Ape-deficient cell lines differentially expressing ref1 or DNA repair activities and characterize these lines with respect to DNA repair capacity and spontaneous mutagenesis and also for AP-1 or NFkB activity. The interaction of the Ape protein with DNA polymerase beta and other proteins (detected by the yeast two-hybrid system or phage display) will be characterized.
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