The Role of Artemis in Cellular Responses to DNA Damage

描述(由申请人提供)：Artemis最近被证明与一种人类严重联合免疫缺陷综合症(SCID)有关，在这种综合症中，观察到T和B细胞缺乏发育。这一缺陷是由于在V(D)J重组的编码联合形成步骤中对Artemis的要求。此外，患者细胞系对电离辐射高度敏感，这表明Artemis也参与了细胞对DNA损伤的反应。本申请的目的是描述Artemis在调节细胞对DNA损伤的反应中所起的作用。具体地说，我们将确定Artemis与之相互作用的蛋白质的性质，并检查其在遗传毒物之前的细胞定位和暴露。我们的发现表明，当细胞暴露于DNA损伤时，Artemis迅速被磷酸化，包括IR和UV引起的DNA损伤。我们还表明，这种磷酸化是由PI3激酶DNA-PK、ATM和ATR介导的。我们将表征Artemis的这种磷酸化，并制备磷酸特异性抗体，用于Artemis的功能研究。这些功能研究将在Artemis缺陷细胞上进行，以确定其在DNA修复和/或细胞周期检查点途径中的作用。这些研究有望阐明Artemis在DNA损伤反应通路中的功能，从而为RS-SCID综合征的放射敏感性提供解释。最后，我们将在小鼠体内准备一个在ATM介导的磷酸化位点发生突变的Artemis敲入突变体。本实验旨在确定ATM对Artemis的修饰是否与AT患者观察到的免疫缺陷有关。

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.