Endo- and exogenous reactive oxygen species (ROS) are intimately involved in aging/senescence. In addition to their role in gene activation, these are invariable genotoxic, and induce a multitude of mutagenic and toxic lesions in in both nuclear and mitochondrial genomes. The DNA damage is repaired via the base excision repair (BER) pathway involving AP-endonuclease (APE). The major mammalian APE (called APE/Ref-1), the rate-linimting enzyme in BER of nuclear DNA lesions, is also responsible for reductive activation of AP-1, a major transcription factor, and may act as a repressor of parathyroid hormone and other genes including its own. Paradoxically, APE/Fer-1 gene is activated by ROS, leading to enhanced repair of oxidative DNA damage. Whether the mitochondrial APE (APE-m), presumably involved in mitochondrial DNA repair, is also activated by ROS. The broad objective of this project is to identify the linkage between age-dependent and ROS- regulated expression of APE/Ref-1 (and possible APE-m) with repair of ROS-induced damage of nuclear and mitochondrial genomes. Using rat and mouce tissues and primary human fibroblasts, a systematic study will be carried out to identify various parameters that affect APE expression. Specifically, the project will: (1) test the hypothesis that ROS activation of APE/Ref-1 as well as its basal levle are APE/Ref-1 levle are secondary to the changes in stress-responsive transcription factors. The roles of glutathione that prevents ROS effects, and of tyrosine phosphorylation of signalling intermediates, will be examine dusing appropriate transgenic mouse mutants. This project will also (4) test the hypothesis that expression of mitochondrial APE-m is O2 in APE activation and BER. Superoxide dismutase-negative mice will be used to examine the role of tested by (5) determining the effect of caloric restruction on age-dependent changes in APEs. The ruslts from this study are expected to provide a significant understanding of the complex andinterlocking fucntions of ROS in DNA damage and its repair, and could suggest approaches to reduce the level of persistent DNA dmanage during aging/senescence.
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