Mechanism and regulation of DNA end processing in V(D)J recombination and repair

V(D)J重组和修复中DNA末端加工的机制和调控

• 批准号: 8097247
• 负责人: JoAnn Sekiguchi
• 金额: $ 34.54万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097247
• 关键词: Alleles; Antigen Receptors; Antigens; Arts; B-Lymphocytes; Binding; Biological Models; Catalytic Domain; Cells; Chromosomal translocation; Code; Codon Nucleotides; Complex; DNA; DNA Double Strand Break; DNA Repair; DNA-dependent protein kinase; Defect; Deoxyribonucleases; Development; Disease; Double Strand Break Repair; Event; Gene Rearrangement; Genes; Genetic; Genome Stability; Genomic Instability; Goals; Human; Immune system; Immunodeficiency and Cancer; Immunologic Deficiency Syndromes; Individual; Inherited; Investigation; J segment gene; Knock-in Mouse; Lead; Ligation; Lymphocyte; Lymphoid; Maintenance; Malignant lymphoid neoplasm; Mediating; Molecular; Mus; Mutation; Nonhomologous DNA End Joining; Nonsense Codon; Nonsense Mutation; Oncogenic; Patients; Pharmaceutical Preparations; Phenotype; Play; Predisposition; Process; Radiation Tolerance; Reading; Receptor Gene; Regulation; Role; ST5 gene; Severe Combined Immunodeficiency; Severities; Syndrome; System; Systems Development; T-Lymphocyte; Testing; Therapeutic; Translations; V(D)J Recombination; artemis; development of lymphoid malignancy; effective therapy; endonuclease; human disease; in vivo; insight; mouse model; mutant; novel; novel therapeutics; nuclease; premature; public health relevance; receptor; recombinational repair; repaired; therapeutic effectiveness; tumor; tumorigenesis; variable region gene

DESCRIPTION (provided by applicant): B and T lymphocytes, the primary cells of the adaptive immune system, provide a major line of defense against a myriad of foreign molecules by virtue of antigen-specific receptors. The vast diversity amongst the antigen receptors genes is generated through V(D)J recombination, a process in which individual V, D, and J gene segments are rearranged. Defects in V(D)J recombination lead to combined immunodeficiencies in human patients and can also result in lymphoid malignancies. One factor that plays a critical role during V(D)J recombination is the non-homologous end joining (NHEJ) DNA repair factor, Artemis. Artemis was initially discovered as the gene inactivated in a human radiosensitive severe combined immunodeficiency syndrome. Hypomorphic Artemis alleles have also been identified in patients and are associated with combined immunodeficiencies of varying severity. Artemis plays a vital role as a DNA nuclease that processes broken ends during V(D)J recombination as well as general double strand break repair prior to ligation. Its intrinsic exo- and endonucleolytic activities are modulated by interaction with the DNA-dependent protein kinase catalytic subunit NHEJ factor. However, the precise mechanisms by which Artemis activities are regulated in vivo are not well understood. The major goals of this proposal are to gain a better understanding of the mechanisms and regulation of DNA end processing during V(D)J rearrangements and general DNA repair. Three specific aims are proposed. Aim 1 is to define the molecular mechanisms involved in activating and regulating Artemis endonucleolytic activities. Aim 2 is to elucidate the molecular mechanisms underlying tumorigenesis caused by a hypomorphic, Artemis disease allele, P70, using the mouse as a model system. This premature translation termination mutation leads to partial immunodeficiency and predisposition to lymphoid malignancy in human patients. In addition, the genetic interactions between Artemis, ATM and Mre11 will be examined. Aim 3 will capitalize on the previously developed Artemis-P70 mouse model, which provides a valuable in vivo system to define the impact of therapeutics that allow read-through of nonsense mutations. The impact of this emerging class of drugs on the DNA repair, genome instability, V(D)J recombination, and lymphocyte development phenotypes in mutant cells and mice will be determined. Together, these studies will provide important insights into the molecular events that occur during V(D)J coding end joining and general DSB repair, elucidate the impact of aberrant V(D)J end processing on lymphocyte development and tumor predisposition, and potentially identify novel therapeutics for primary immunodeficiencies caused by nonsense mutations. PUBLIC HEALTH RELEVANCE: This proposal will examine the molecular mechanisms and regulation of DNA end processing during V(D)J recombination and the consequences of aberrant end processing on immune system development and lymphoid malignancies. In addition, the impact of premature termination codon read-through therapeutics on treating immunodeficiency, DNA repair and genome instability phenotypes using a unique mouse model will be examined. Together, these studies will not only provide important insights into the basic mechanisms of V(D)J recombination, but also lead to a better understanding of the in vivo consequences of specific human disease alleles as well as potentially identify novel therapeutics for inherited primary immunodeficiencies.

描述（由申请人提供）：B和T淋巴细胞是适应性免疫系统的主要细胞，通过抗原特异性受体提供对抗无数外来分子的主要防线。抗原受体基因之间的巨大多样性是通过V（D）J重组产生的，V（D）J重组是单个V、D和J基因片段重排的过程。V（D）J重组缺陷导致人类患者的联合免疫缺陷，也可能导致淋巴恶性肿瘤。在V（D）J重组过程中起关键作用的一个因子是非同源末端连接（NHEJ）DNA修复因子Artemis。Artemis最初是在人类放射敏感性严重联合免疫缺陷综合征中发现的失活基因。亚型Artemis等位基因也已在患者中鉴定，并与不同严重程度的联合免疫缺陷相关。Artemis作为一种DNA核酸酶在V（D）J重组过程中处理断裂末端以及连接前的一般双链断裂修复中起着至关重要的作用。其内在的外切和内切核酸裂解活性通过与DNA依赖性蛋白激酶催化亚基NHEJ因子的相互作用来调节。然而，Artemis活动在体内调节的确切机制还不清楚。 本研究的主要目的是为了更好地理解V（D）J重排和一般DNA修复过程中DNA末端加工的机制和调控。提出了三个具体目标。目的1是明确参与激活和调节Artemis核酸内切酶活性的分子机制。目的2是阐明潜在的肿瘤发生的分子机制，由一个亚型，阿耳忒弥斯病等位基因，P70，使用小鼠作为模型系统。这种过早的翻译终止突变会导致人类患者出现部分免疫缺陷和淋巴恶性肿瘤的易感性。此外，还将研究Artemis、ATM和Mre 11之间的遗传相互作用。目标3将利用先前开发的Artemis-P70小鼠模型，该模型提供了一个有价值的体内系统来定义允许无义突变通读的治疗方法的影响。将确定这类新兴药物对突变细胞和小鼠中DNA修复、基因组不稳定性、V（D）J重组和淋巴细胞发育表型的影响。总之，这些研究将为V（D）J编码末端连接和一般DSB修复过程中发生的分子事件提供重要见解，阐明异常V（D）J末端加工对淋巴细胞发育和肿瘤易感性的影响，并可能确定无义突变引起的原发性免疫缺陷的新疗法。 公共卫生关系：本提案将研究V（D）J重组过程中DNA末端加工的分子机制和调控，以及异常末端加工对免疫系统发育和淋巴恶性肿瘤的影响。此外，将使用独特的小鼠模型检查提前终止密码子通读疗法对治疗免疫缺陷、DNA修复和基因组不稳定性表型的影响。总之，这些研究不仅将为V（D）J重组的基本机制提供重要的见解，而且还将更好地理解特定人类疾病等位基因的体内后果，并可能确定遗传性原发性免疫缺陷的新疗法。