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The Role of Artemis in Cellular Responses to DNA Damage

Artemis 在细胞对 DNA 损伤反应中的作用

基本信息

批准号：
7394440
负责人：
RANDY J LEGERSKI
金额：
$ 29.35万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-03-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7394440
关键词：
1-Phosphatidylinositol 3-Kinase Affect Alleles Animals Asses Ataxia Telangiectasia B-Lymphocytes Binding Biological Assay Cell Cycle Arrest Cell Cycle Checkpoint Cell Line Cells Co-Immunoprecipitations Code Complex DNA Damage DNA Repair DNA-dependent protein kinase Defect Development Double Strand Break Repair Exhibits Exposure to Failure Gel Chromatography Genes Goals Homologous Gene Human Human Cell Line Immune Immunologic Deficiency Syndromes In Vitro Ionizing radiation Joints Knock-in Mouse Macromolecular Complexes Mammalian Cell Mechlorethamine Mediating Mediation Medical Surveillance Methods Modification Mus Mutagens Mutate Nature Nonhomologous DNA End Joining Pathway interactions Patients Phenotype Phospho-Specific Antibodies Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide Phosphorylation Phosphorylation Site Phosphotransferases Play Proteins Radiation Tolerance Resistance Role Saccharomyces cerevisiae Severe Combined Immunodeficiency Signal Transduction Site Syndrome Testing V(D)J Recombination Yeasts artemis crosslink design gene function in vivo mutant protein protein interaction research study response yeast two hybrid system

项目摘要

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.

描述（由申请人提供）：Artemis最近被证明与人类严重联合免疫缺陷综合征（SCID）有关，其中观察到T细胞和B细胞缺乏发育。这种缺陷是由于在V(D)J重组的编码接头形成步骤中需要Artemis。此外，患者细胞系显示对电离辐射过敏，这表明Artemis也参与细胞对DNA损伤的反应。本应用的目的是表征Artemis在介导细胞对DNA损伤反应中的作用。具体来说，我们将确定与Artemis相互作用的蛋白质的性质，并检查其在暴露于遗传毒性物质之前的细胞定位。我们的研究结果表明，当细胞暴露于包括红外和紫外线诱导的DNA损伤时，Artemis会迅速磷酸化。我们还发现这种磷酸化是由PI3激酶DNA-PK、ATM和ATR介导的。我们将对Artemis的磷酸化进行表征，并制备磷酸化特异性抗体，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。期望这些研究能够阐明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，我们将在小鼠中制备Artemis的敲入突变体，该突变体在ATM介导的磷酸化位点发生突变。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。