FANCONI ANEMIA GENE PATHWAY IN RADIATION RESPONSES

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The project's objective is to understand the molecular regulatory processes cells use to minimize genetic damage and genetic instability associated with reactive oxygen species (ROS) arising from endogenous processes or ionizing radiation (IR). This goal is addressed through studies of FANCG/XRCC9, the gene that is defective in. group G of the cancer-prone disorder Fanconi anemia (FA). Because FancG protein confers IR resistance in hamster cells, the human homolog is expected to participate in IR responses in human cells. Historically, a link between the FA genes and radiation responses has been unclear, with some studies suggesting that the primary defect in FA lies in removing DNA interstrand crosslinks. The general hypothesis to be tested is that the FANCG protein, as a member of a multiprotein complex, protects mammalian cells against endogenous and IR-generated oxidative damage and maintains genomic integrity by coordinating homeostasis processes that include regulation of ROS levels, apoptosis, and cell cycle progression. The proposed studies will provide a highly quantitative characterization of FANCG protein's contribution to biochemical and cellular endpoints associated with both normal cell proliferation and responses to IR exposure. Isogenic pairs of mutant and FANCG-complemented cells will be derived in both hamster CHO cells and human lymphoblasts. These pairs will be analyzed with respect to chromosomal aberrations, cell survival, hprt gene mutations, apoptosis, ROS, and cell cycle parameters with and without IR exposure. The FANCG-complemented FA-G lymphoblasts will be used to examine gene and protein regulation during the cell cycle as well as the subcellular localization of the protein with and without IR damage. Three proteins that are candidate interactors with FANCG from preliminary studies will be evaluated for possible involvement in the FA pathway. Finally, already identified high-frequency human allelic variants of FANCG in the US population will be evaluated for degree of dysfunction. The results of these studies will lead to more specific models of the nature of the FA protein "pathway" and its quantitative contributions to multiple biological effects associated with IR-mediated oxidative damage.