p53 in Cellular Response to ROS-Mediated DNA Damage

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DESCRIPTION (provided by applicant): The tumor suppressor p53 plays important roles in regulating gene expression, cell cycle progression, and apoptosis in response to DNA damage. Our studies on the role of p53 in repair of drug-induced DNA damage, supported by a NIH R29 grant, have led to several important findings. We demonstrated that the 3'- 5' exonuclease activity of p53 preferentially removed mismatched nucleotides from DNA and enhanced DNA replication fidelity in vitro, suppressed mismatch mutations in whole cells, and may function as a sensor component for drug-induced DNA damage. Importantly, we observed that p53 was activated by anticancer agents that cause accumulation of reactive oxygen species (ROS), interacted with a repair enzyme APE/Ref-1 in binding to ROS-damaged DNA, and triggered apoptosis. This p53 activation was significantly affected by the mitochondrial respiratory activity. These observations, together with the facts that mitochondria play a major role in ROS generation and redox regulation, suggest a logical link between the mitochondrial respiration and p53 activation in sensing ROS-mediated DNA damage and causing cell death. The long-term goals of this research project are to investigate the roles of p53 and mitochondria in cellular response to ROS-mediated DNA damage and drug-induced apoptosis, and to evaluate their relevance in cancer therapeutics. We will use biochemical and molecular biology methods to investigate the following specific aims: (1) Test the hypothesis that p53 functions as a component of a sensor complex for ROS-damaged DNA, is activated during interaction with the damaged DNA, and trigger apoptosis when DNA damage is persistent. In vitro assays using isolated protein components and DNA containing defined oxidative damage will be employed to test the physical and functional interactions between p53 and base excision repair molecules such as 8- oxoguanine glycosylase and AP endonuclease. (2) Characterize the novel role of mitochondrial respiration on p53 activation during cellular response to ROS-mediated DNA damage, and investigate the underlying mechanisms. Cell lines with various p53 genotypes and with genetically altered mitochondria deficient in respiration have been established in our laboratory as unique tools for these studies. (3) Evaluate the biological consequences of 53 activation in response to oxidative DNA damage caused by ROS and relevant anticancer agents. Isogenic p53 cell lines will be tested for differential sensitivity to ROS-generating agents. It is anticipated that the proposed studies will further our understanding of the mechanisms by which p53 affect cellular response to oxidative DNA damage and sensitivity to anticancer agents that generate ROS.