Effect of VDJ Recombination on Scid B Cell Development

淋巴系统恶性肿瘤的形成与一个基本的生理过程有关。 淋巴细胞发育，V(D)J重组。这种重组通过两个步骤进行：一个站点- 特定的切割产生双链DNA断裂，随后对这些断裂进行一般修复。这个 后者由非同源末端连接(NHEJ)蛋白进行。NHEJ基因的突变已经被 显示极大地增加了基因组的不稳定性和淋巴细胞的致癌转化。这一发现不是 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 另一种末端连接途径的异常性质，可能导致基因异常。然而，几乎没有 已知V(D)J重组中这一异常途径的分子基础以及随后的遗传 改装。 我们的长期目标是阐明V(D)J缺陷重组的潜在机制 淋巴细胞致癌转化的过程和识别可能增强的潜在危险因素 这些过程。为了实现这一目标，我们开发了重组诱导细胞系。 NHEJ-突变体，一种严重的联合免疫缺陷(SCID)小鼠，其催化单位存在突变 DNA依赖蛋白激酶(DNA-PKcs)。大量的重组中间体可以被 在这种SCID细胞系中产生的，其分辨率是有条件的，容易出错，并导致形成 基因座间重组产物。我们建议扩展我们在DNA-PKcs非依赖性方面的工作 通过开发一种新的方法来捕获任何经历重组诱导的细胞进行重组 易位。为了进一步了解SCID细胞异常末端分辨率的生化性质，我们 将表征与分解过程中的重组末端相关的蛋白质结构，以及 直接检测外源表达的重组酶对SCID编码末端分辨率的作用。最后，我们 将研究聚(ADP-核糖基)作用，几种DNA损伤传感系统之一，在 在这些SCID细胞中识别、处理和连接重组末端，以及维持基因组 稳定性。

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.