TdT and pol mu in DNA repair and immune system diversity

描述(申请人提供)：哺乳动物非同源末端连接(NHEJ，End Join)途径在所有类型的细胞中用于修复DNA损伤剂(如电离辐射、化疗药物)引起的DNA双链断裂(DSB)。末端连接对于V(D)J重组过程中DSB中间体的有效分解也是至关重要的，V(D)J重组是一种淋巴特有的过程，需要组装免疫系统的抗原特异性受体。有缺陷的末端连接因此会导致放射敏感性、癌症发病率增加以及免疫缺陷。两个相关的DNA聚合酶，淋巴特异的末端脱氧核苷酸转移酶(TDT)和最近描述的聚合酶MU(PolMU)，与末端连接所需的因子特异性地相关。 1)Polu在末端连接中的作用尚不清楚。细胞V(D)J重组分析将被用来阐明polu在V(D)J重组中的作用，以及它在末端连接修复中的作用。 2)以前的细胞实验已经概述了聚合酶在末端连接中的特征活性，因此这一途径比预期的更准确。使用纯化因子的简化系统将用于确定Polu、TdT或其他聚合酶是否具有这些特征活性。POLMU或TDT中的目标突变也将被进行，以更好地理解这些特定聚合酶与末端连接因子相关联的能力在实现更准确的末端连接方面的重要性。 3)TdT和Polu在体外合成过程中很容易同时结合DNA和RNA，而所有其他已知的核酸聚合酶(RNA或DNA)通常结合适当的核酸类型的效率至少是不适当的核酸类型的1000倍。实验将确定是否也可以在细胞中的V(D)J重组(或末端连接DSB修复)位置观察到这种活性，以及这些聚合酶在体外对末端连接的RNA掺入有什么影响。 这项工作将提供一个全面的了解聚合酶在特定的，和一般的末端处理因素，是如何被末端连接途径用于DSB修复，从而使这种修复途径是准确的。它将有助于解决如何控制这些加工因子的活性，如何在可接受的风险下实现最终加工，以及不受控制的加工活动可能对基因组稳定性造成的后果。

DESCRIPTION (provided by applicant): The mammalian non-homologous end joining (NHEJ, end joining) pathway is employed in all cell types to repair DNA double strand breaks (DSBs) caused by DNA damaging agents (e.g. ionizing radiation, chemotherapeutic drugs). End joining is also essential for efficient resolution of DSB intermediates during V(D)J recombination, a lymphoid specific process required to assemble the immune system's antigen specific receptors. Defective end joining thus results in radiosensitivity, an increased incidence of cancer, as well as immunodeficiency. Two related DNA polymerases, the lymphoid-specific Terminal deoxynucleotidyl transferase (TdT) and the recently described polymerase mu (pol mu), specifically associate with factors required for end joining. 1) The role of pol mu in end joining is as yet unclear. Cellular V(D)J recombination assays will be used to clarify pol mu's role in V(D)J recombination, as well as its role in end joining repair in general. 2) Previous cellular experiments have outlined characteristic activities of polymerases in end joining, such that this pathway is more accurate than would be expected otherwise. A reduced system, using purified factors, will be used to determine if pol mu, TdT, or other polymerases possess these characteristic activities. Target mutations in pol mu or TdT will also be made to better understand the importance of the ability of these specific polymerases to associate with end joining factors in achieving more accurate end joining. 3) TdT and pol mu readily incorporate both DNA and RNA during synthesis in vitro, while all other known nucleic acid polymerases (RNA or DNA) typically incorporate the appropriate nucleic acid type at least 1000 times more efficiently than the inappropriate nucleic acid type. Experiments will be performed do determine if this activity can also be observed at sites of V(D)J recombination (or end joining DSB repair) in cells, and what impact RNA incorporation by these polymerases has on end joining in vitro. This work will provide a comprehensive understanding how polymerases in specific, and end processing factors in general, are employed by the end joining pathway for DSB repair such that this repair pathways is accurate. It will help address how the activity of these processing factors are controlled, how end processing is achieved with acceptable risk, and what the consequences of un-controlled processing activity might be to genome stability.