The Role of Artemis in Cellular Responses to DNA Damage

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DESCRIPTION (provided by applicant): Artemis has been shown recently to be involved in a human severe combined immunodeficiency syndrome (SCID) in which an absence of development of T and B cells is observed. This defect is due to a requirement for Artemis in the coding joint formation step of V(D)J recombination. In addition, patient cell lines were shown to be hypersensitive to ionizing radiation suggesting that Artemis is also involved in cellular responses to DNA damage. The goals of this application are to characterize the function of Artemis in regard to its role in mediating the cellular response to DNA damage. Specifically, we will determine the nature of proteins that Artemis interacts with, and examine its cellular localization before and exposure to genotoxic agents. Our findings show that Artemis is rapidly phosphorylated upon exposure of cells to DNA damage including that induced by both IR and UV. We have also shown that this phosphorylation is mediated by the PI3 kinases DNA-PK, ATM, and ATR. We will characterize this phosphorylation of Artemis and prepare phosphospecific antibodies that will be utilized for functional studies of Artemis. These functional studies will be conducted on cells defective in Artemis to determine its role in either DNA repair and/or cell cycle checkpoint pathways. It is expected that these studies will elucidate the function of Artemis in DNA damage response pathways, and thus provide an explanation for the radiosensitivity of the RS-SCID syndrome. Finally, we will prepare a knock-in mutant of Artemis in the mouse that is mutated at sites of phosphorylation mediated by ATM. This experiment is designed to determine if the modification of Artemis by ATM is involved in the immunodeficiency observed in AT patients.