The Role of Artemis in Cellular Responses to DNA Damage

Artemis 在细胞对 DNA 损伤反应中的作用

• 批准号: 7026429
• 负责人: RANDY J LEGERSKI
• 金额: $ 30.23万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7026429
• 关键词: DNA damage; DNA repair; cell cycle; cell growth regulation; cell line; cell proliferation; clinical research; functional /structural genomics; gel filtration chromatography; gene expression; gene interaction; genetic recombination; genetic regulation; genetically modified animals; immunocytochemistry; immunoprecipitation; intermolecular interaction; laboratory mouse; phosphorylation; radiation sensitivity; radiobiology; severe combined immunodeficiency; yeast two hybrid system

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和B细胞发育缺失。该缺陷是由于在V（D）J重组的编码联合形成步骤中需要Artemis。此外，患者细胞系显示对电离辐射过敏，表明Artemis也参与了对DNA损伤的细胞反应。本申请的目的是表征Artemis在介导细胞对DNA损伤的反应方面的功能。具体来说，我们将确定与Artemis相互作用的蛋白质的性质，并检查其细胞定位之前和暴露于遗传毒性剂。我们的研究结果表明，Artemis是迅速磷酸化后暴露的细胞DNA损伤，包括诱导的IR和UV。我们还表明，这种磷酸化是由PI 3激酶DNA-PK，ATM和ATR介导的。我们将表征这种Artemis的磷酸化，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损伤反应途径中的作用，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介导的磷酸化位点处突变。本实验旨在确定ATM对Artemis的修饰是否涉及AT患者中观察到的免疫缺陷。