Artemis: Biochemical, structural and functional analysis

在淋巴细胞发育过程中，抗原受体基因由种系V、D和J基因组装而成 片段通过称为V(D)J重组的特定部位的体细胞重组过程。这里面的瑕疵 途径导致许多原发免疫缺陷，如严重联合免疫缺陷(SCID) 和Omenn综合征，并可能导致白血病和淋巴瘤的发展。两个淋巴组织 特定的蛋白质RAG1和RAG2将DNA切割在编码片段和信号序列之间。这个 非同源末端连接(NHEJ)DNA修复途径修复由此产生的双链断裂。 该反应导致编码末端具有共价闭合的发夹结构，需要打开和 在结扎前处理。一种名为Artemis的新蛋白的突变导致RS-SCID综合征，一种SCID 与辐射敏感性相关的表型。来自体外分析和基因敲除小鼠的数据表明 Artemis是打开发夹编码端的核酸酶。然而，人们对生物化学一无所知。 以及这种蛋白质的结构。此外，仍然缺乏其在NHEJ中所起作用的直接证据。另外，C端子 构成几乎一半蛋白质的Artemis区域尚未被研究。在这项拨款申请中，我们 描述一系列实验，以解决这些问题，同时扩大我们的观察，阿尔特弥斯 在体内通过其C-末端结构域与Ligase IV/XRCC4相互作用，该复合体在DSB中非常活跃 体外结扎。通过提出的具体目标，我们将1)表征生物化学特性 Artemis及其晶体结构的发展；2)研究Artemis与其相互作用的体内相关性 NHEJ中的连接酶IV，并确定其C-末端区域在NHEJ中的作用，以及3)确定这一意义 在V(D)J重组中的相互作用，并确定其C-末端区域的作用。总之，这些实验 为具体目标提出的建议将开始界定Artemis的结构-功能关系和 在人类中发现不同RS-SCID突变的机制。此外，通过确立 Artemis与Ligase IV在V(D)J重组和体内NHEJ中的相互作用，我们将提供新的 对阿尔特弥斯在这些途径中扮演的多重角色的洞察。通过这项研究，我们还将描述一种 新的蛋白质结构，通过求解Artemis的蛋白质结构，进一步确定其在发育中的作用 健康的免疫系统。

During lymphocyte development, antigen receptor genes are assembled from germline V,D and J gene segments by a site-specific somatic recombination process termed as V(D)J recombination. Defects in this pathway result in a number of primary immunodeficiencies like Severe Combined Immuno Deficiency (SCID) and Omenn syndrome and potentially can lead to development of leukemias and lymphomas. Two lymphoid specific proteins RAG1 and RAG2 cleave DNA between the coding segments and signal sequences. The Non-Homologous End Joining (NHEJ) DNA repair pathway repairs the double strand breaks thus generated. This reaction results in coding ends with covalently closed hairpin structures that need to be opened and processed before ligation. Mutations in a novel protein called Artemis causes RS-SCID syndrome, a SCID phenotype associated with radiosensitivity. Data from in vitro analysis and knockout mice have shown that Artemis is the nuclease that opens hairpin-coding ends. However, nothing is known about the biochemistry and structure of this protein. Further, direct evidence for its role in NHEJ is still lacking. Also, the C-terminal region of Artemis that forms almost half of the protein has not been studied. In this grant application, we describe a series of experiments that will address these issues while extending our observation that Artemis interacts with Ligase IV/XRCC4 in vivo through its C-terminal domain and this complex is very active in DSB ligation in vitro. Through specific aims proposed we will 1) characterize the biochemical properties of Artemis and develop its crystal structure, 2) investigate the in vivo relevance of the interaction of Artemis with Ligase IV in NHEJ and define a role of its C-terminal region in NHEJ and 3) establish the significance of this interaction in V(D)J recombination and ascertain the role of its C-terminal region. Together, the experiments proposed for the specific aims will begin to define the structure-function relationship of Artemis and the mechanism of different RS-SCID mutations found in humans. Further, by establishing the importance of interaction of Artemis with Ligase IV in both V(D)J recombination and NHEJ in vivo, we will provide novel insights into multiple roles played by Artemis in these pathways. Through this study, we will also describe a novel protein structure, by solving the protein structure of Artemis and further define its role in development of a healthy immune system.