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

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了一条主要的防线来抵御无数外来分子。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，这是一个单独的V、D和J基因片段重排的过程。V(D)J重组缺陷会导致人类患者的联合免疫缺陷，也可能导致淋巴系统恶性肿瘤。在V(D)J重组过程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。Artemis最初是在一种对辐射敏感的严重联合免疫缺陷综合征中被发现的基因失活。在患者中也发现了低态Artemis等位基因，这些等位基因与不同严重程度的联合免疫缺陷有关。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在连接前进行一般的双链断裂修复，起着至关重要的作用。其内源性和外源性溶核活性受依赖于DNA的蛋白激酶催化亚基NHEJ因子的相互作用调节。然而，Artemis活动在体内被调控的确切机制还不是很清楚。这一建议的主要目的是为了更好地了解V(D)J重排和一般DNA修复过程中DNA末端加工的机制和调节。提出了三个具体目标。目的1确定激活和调节Artemis核酸内切活性的分子机制。目的2是以小鼠为模型系统，阐明由一种亚型Artemis病等位基因P70引起的肿瘤发生的分子机制。这种过早的翻译终止突变会导致人类患者的部分免疫缺陷和淋巴系统恶性肿瘤的易感性。此外，还将研究Artemis、ATM和Mre11之间的遗传交互作用。AIM 3将利用之前开发的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通读无义突变的疗法的影响。这类新兴药物对突变细胞和小鼠的DNA修复、基因组不稳定、V(D)J重组和淋巴细胞发育表型的影响将被确定。综上所述，这些研究将对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的见解，阐明V(D)J末端异常加工对淋巴细胞发育和肿瘤易感性的影响，并可能为无义突变引起的原发免疫缺陷寻找新的治疗方法。 与公共卫生相关：这项建议将研究V(D)J重组过程中DNA末端处理的分子机制和调控，以及异常末端处理对免疫系统发育和淋巴系统恶性肿瘤的影响。此外，还将使用独特的小鼠模型研究提前终止密码子通读疗法在治疗免疫缺陷、DNA修复和基因组不稳定表型方面的影响。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，还将有助于更好地了解特定人类疾病等位基因在体内的后果，并有可能找到治疗遗传性初级免疫缺陷的新疗法。

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.