Checkpoint Signaling and Repair of UV Damage to Human DNA

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DESCRIPTION (provided by applicant): The long-term objectives of this project are to delineate the molecular mechanisms of interpathway interactions between nucleotide excision repair (NER) and DNA damage checkpoints. NER and the damage checkpoints are two major components in cellular responses to DNA damage due to formation of bulky lesions induced by UV irradiation and various environmental genotoxic chemicals and carcinogens. Defects in these pathways have been directly correlated to human cancer prone deceases such as xeroderma pigmentosum (XP) and Ataxia-Telangiectasia (AT). Although great efforts have been made in understanding the overall mechanism of cellular DNA damage responses, the molecular details about the relationship between the two pathways remain elusive due to the fact that the two systems have been largely studied separately. To gain a more systematic and precise view of cellular responses to DNA damage, experiments and strategies are developed to address the following questions: How the DNA damage checkpoints and DNA repair are coordinated in cells upon DNA damage? What are the protein factors involved in the interactions? And what is the molecular basis of the interactions? Using a combined biochemical, molecular biology and structural approach, this project aims (1) to define the role XPA in ATR regulation of NER and the cell-cycle dependency of the regulation for testing the hypothesis that XPA is the main target in NER for regulation of NER functions by ATR checkpoint; (2) to determine the effects of ATR-XPA protein-protein interaction on DNA damage responses to UV and to test the underlying hypothesis that the interaction is a determinant for nuclear accumulation of XPA for NER regulated by ATR upon UV irradiation; (3) to examine the dependence of nuclear-translocation of XPA on ATR checkpoint signaling. We will test the hypothesis that ATR checkpoint signaling is required for the DNA damage-induced cytoplasm-to-nuclear trafficking of XPA; and (4) to assess the role of ATR- regulated XPA phosphorylation in DNA damage responses and the effects of XPA phosphorylation on nucleotide excision repair.