Effect of VDJ Recombination on Scid B Cell Development

描述(申请人提供)：淋巴系恶性肿瘤的形成与淋巴细胞发育的一个基本生理过程--V(D)J重组有关。这种重组通过两个步骤进行：特定位点的切割产生双链DNA断裂，然后对这些断裂进行一般修复。后者由非同源末端连接(NHEJ)蛋白完成。NHEJ基因的突变已被证明极大地增加了基因组的不稳定性和淋巴细胞的致癌转化。这一发现不仅表明NHEJ蛋白在维持基因组完整性方面的重要性，而且还表明了可能引发遗传异常的另一种末端连接途径的异常性质。然而，关于V(D)J重组中这种异常途径的分子基础以及随后的遗传改变，人们知之甚少。我们的长期目标是阐明V(D)J缺陷重组过程在淋巴细胞致癌转化中的潜在机制，并识别可能增强这些过程的潜在危险因素。为了实现这一目标，我们从一个NHEJ突变体中培育了重组可诱导细胞系，该突变体是一种严重的联合免疫缺陷(SCID)小鼠，其DNA依赖蛋白激酶(DNA-PKcs)的催化单元发生突变。在该SCID细胞系中可以产生大量的重组中间体，并且它们的拆分是有条件的，容易出错，并导致基因座间重组产物的形成。我们建议通过开发一种新的方法来捕获任何经历重组诱导易位的细胞，从而扩展我们在DNA-PKcs非依赖性重组方面的工作。为了进一步了解SCID细胞末端分辨异常的生化本质，我们将在分辨过程中表征与重组末端相关的蛋白质结构，并直接检测外源表达的重组酶在SCID编码末端分辨中的功能。最后，我们将研究几种DNA损伤传感系统之一的聚(ADP-核糖化)在识别、处理和连接这些SCID细胞中的重组末端以及维持基因组稳定性方面的作用。

DESCRIPTION (provided by the applicant): The formation of lymphoid malignancies has been linked to an essential physiological process of lymphocyte development, V(D)J recombination. This recombination proceeds through two steps: a site specific cleavage to generate double stranded DNA breaks, followed by a general repair of these breaks. The latter is carried out by non-homologous end joining (NHEJ) proteins. Mutations in NHEJ genes have been shown to greatly increase genomic instability and oncogenic transformation of lymphocytes. This finding not only indicates the importance of NHEJ proteins in maintaining genome integrity, but also suggests the aberrant nature of an alternative end-joining pathway that may initiate genetic abnormalities. However, little is known about the molecular basis of this aberrant pathway in V(D)J recombination and consequent genetic alterations. Our long term objective is to elucidate the underlying mechanisms of defective V(D)J recombination processes in oncogenic transformation of lymphocytes and to identify potential risk factors that may enhance these processes. In an effort to achieve this goal, we developed recombination-inducible cell lines from one NHEJ-mutant, a severe combined immunodeficient (scid) mouse that bears a mutation in the catalytic unit of the DNA-dependent protein kinase (DNA-PKcs). A large quantity of recombination intermediates can be generated in this scid cell line, and their resolution is conditional, error-prone, and leads to the formation of interlocus recombination products. We propose to extend our work on DNA-PKcs-independent recombination by developing a novel approach to capture any cells that undergo recombination-induced translocation. To further understand the biochemical nature of the aberrant end resolution in scid cells, we will characterize protein structures associated with recombination ends during the course of resolution, and directly test the function of exogenously expressed recombinase on scid coding end resolution. Finally, we will investigate the role of poly(ADP-ribosyl)ation, one of several DNA damage sensing systems, in recognizing, processing and joining recombination ends in these scid cells, as well as in maintaining genome stability.