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）：自适应免疫系统的主要细胞B和T淋巴细胞通过抗原特异性受体提供了针对无数外国分子的主要防御方法。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，即单个V，D和J基因段的重组。 V（D）J重组中的缺陷导致人类患者的免疫缺陷，也可能导致淋巴恶性肿瘤。在V（D）重组中起关键作用的一个因素是非同源末端连接（NHEJ）DNA修复因子Artemis。最初发现Artemis是在人类放射敏感的严重合并免疫缺陷综合征中灭活的基因。还已经在患者中鉴定出肌肉症等位基因，并且与严重程度不同的免疫缺陷相关。 Artemis作为DNA核酸酶起着至关重要的作用，该核酸酶在连接之前在V（d）J重组以及一般的双链断修复过程中处理破裂的末端。它的固有外核和内核液化活性是通过与DNA依赖性蛋白激酶催化亚基NHEJ因子的相互作用来调节的。但是，在体内调节Artemis活动的确切机制尚不清楚。 该提案的主要目标是更好地了解V（d）J重排和一般DNA修复期间DNA终端加工的机制和调节。提出了三个具体目标。目的1是定义激活和调节artemis内核酸化活性的分子机制。目的2是使用小鼠作为模型系统，阐明由肌肉病等位基因，p70 p70引起的肿瘤发生的分子机制。这种过早的翻译终止突变导致人类患者的淋巴性恶性肿瘤的部分免疫缺陷。此外，将检查Artemis，ATM和MRE11之间的遗传相互作用。 AIM 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，以定义允许读取无意义突变的治疗剂的影响。这种新兴药物对突变细胞和小鼠在突变细胞和小鼠中的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，这些研究将提供对V（d）J编码端和一般DSB修复过程中发生的分子事件的重要见解，阐明了异常v（d）J末端加工对淋巴细胞发育和肿瘤易感性的影响，并潜在地鉴定出对原发性免疫缺陷引起的新型治疗方法，导致无态突变引起的原发性免疫缺陷。 公共卫生相关性：该提案将检查V（d）J重组期间DNA终端加工的分子机制和调节，以及异常终端加工对免疫系统发育和淋巴恶性肿瘤的后果。此外，将检查使用独特的小鼠模型的过早终止密码子读取治疗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，这些研究不仅将提供对V（d）J重组的基本机制的重要见解，而且还可以更好地理解特定人类病等位基因的体内后果，并有潜在地鉴定出对遗传性原发性免疫缺陷的新型治疗剂。