Mutagenic and toxic abasic sites (APS) and their oxidized derivatives (OAS), implicated in induction of sporadic cancer, are endogenously generated in the nuclear and mitochondrial genomes of mammalian cells by reaction with reactive oxygen species (ROS) produced in the mitochondria. Ubiquitous AP-endonuclease (APE), an endonuclease/3' phosphodiesterase, is responsible for the repair of APS and most OAS. The mammalian genome, unlike E. coli or yeast, encodes only one APE (APE1) which has additional regulatory activities. Inactivation of APE1 is lethal for mouse embryos, and no APE1 null cells can be isolated. During our current funding period, we established conditional APE1 null mouse embryo fibroblast (MEF) lines in which deletion of the APE1 transgene induces apoptosis that is prevented by ectopic APE1 containing both repair and regulatory activities. We hypothesize that APE1 is essential because mitochondrial (mt) APE, resulting from its N-terminal cleavage by an unknown ROS-activated APE1 protease (RAAP), repairs APS and OAS in the mt genome. In the absence of repair, ROS at increasing levels induce APS and OAS in the nuclear genome, and activate signaling for mt-regulated apoptosis. We will test this broad hypothesis by pursuing the following Specific Aims, to; (1) generate transfectants of APE1 conditional MEF mutants for stable expression of APEI's regulatory function in order to exclusively examine APE1's repair activity for preventing apoptosis. (2) test the hypothesis that APS and OAS in the mt genome provide the initial ROS signal for apoptosis by measuring ROS and the kinetics of APS and OAS formation in the absence of APE1; (3) purify and clone RAAP in order to show that its inhibition prevents APE1's cleavage and hence mt accumulation, leading to apoptosis, and finally (4) test the hypothesis that the nuclear APS and OAS activate both p53 and p73 for inducing apoptosis. The mutant MEF system will uniquely allow us to elucidate the etiological role of endogenous DNA damage in the nuclear vs. mt genomes, in cellular toxicity and carcinogenic transformation, and measure their true rate of generation. Health relevance: This project will explore the reasons why the oxidative damage repair activity of APE1 is essential in mammalian cells. In view of APE1's frequent overexpression in tumor cells, the results of our project could help design novel strategies involving identification of therapeutic targets for preventing sporadic cancer as well as improving its treatment. ? ? ?

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Okano, Paul
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University of Texas Medical Br Galveston
Schools of Medicine
United States
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Suganya, Rangaswamy; Chakraborty, Anirban; Miriyala, Sumitra et al. (2015) Suppression of oxidative phosphorylation in mouse embryonic fibroblast cells deficient in apurinic/apyrimidinic endonuclease. DNA Repair (Amst) 27:40-8
Szczesny, Bartosz; Brunyanszki, Attila; Olah, Gabor et al. (2014) Opposing roles of mitochondrial and nuclear PARP1 in the regulation of mitochondrial and nuclear DNA integrity: implications for the regulation of mitochondrial function. Nucleic Acids Res 42:13161-73
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Szczesny, Bartosz; Olah, Gabor; Walker, Dillon K et al. (2013) Deficiency in repair of the mitochondrial genome sensitizes proliferating myoblasts to oxidative damage. PLoS One 8:e75201
Hegde, Muralidhar L; Izumi, Tadahide; Mitra, Sankar (2012) Oxidized base damage and single-strand break repair in mammalian genomes: role of disordered regions and posttranslational modifications in early enzymes. Prog Mol Biol Transl Sci 110:123-53
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Mantha, Anil K; Dhiman, Monisha; Taglialatela, Giulio et al. (2012) Proteomic study of amyloid beta (25-35) peptide exposure to neuronal cells: Impact on APE1/Ref-1's protein-protein interaction. J Neurosci Res 90:1230-9
Sengupta, Shiladitya; Chattopadhyay, Ranajoy; Mantha, Anil K et al. (2012) Regulation of mouse-renin gene by apurinic/apyrimidinic-endonuclease 1 (APE1/Ref-1) via recruitment of histone deacetylase 1 corepressor complex. J Hypertens 30:917-25
Hegde, Muralidhar L; Hegde, Pavana M; Arijit, Dutta et al. (2012) Human DNA Glycosylase NEIL1's Interactions with Downstream Repair Proteins Is Critical for Efficient Repair of Oxidized DNA Base Damage and Enhanced Cell Survival. Biomolecules 2:564-78

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