TdT and pol mu in DNA repair and immune system diversity

描述（由申请人提供）：哺乳动物非同源末端连接（NHEJ，末端连接）途径在所有细胞类型中用于修复由DNA损伤剂（例如电离辐射、化疗药物）引起的DNA双链断裂（DSB）。 末端连接对于V（D）J重组期间DSB中间体的有效解析也是必不可少的，V（D）J重组是组装免疫系统的抗原特异性受体所需的淋巴特异性过程。 因此，末端连接缺陷导致放射敏感性、癌症发病率增加以及免疫缺陷。 两种相关的DNA聚合酶，淋巴特异性末端脱氧核苷酸转移酶（TdT）和最近描述的聚合酶μ（pol μ），专门与末端连接所需的因子。 1)pol mu在末端连接中的作用尚不清楚。 细胞V（D）J重组测定将用于阐明pol mu在V（D）J重组中的作用，以及其在一般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，使得该途径比预期的更准确。 使用纯化因子的还原系统将用于确定pol mu、TdT或其他聚合酶是否具有这些特征活性。 还将进行pol mu或TdT中的靶突变，以更好地理解这些特异性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中的重要性。 3)TdT和pol mu在体外合成过程中容易掺入DNA和RNA，而所有其他已知的核酸聚合酶（RNA或DNA）通常掺入适当的核酸类型的效率比不适当的核酸类型高至少1000倍。 将进行实验以确定这种活性是否也可以在细胞中的V（D）J重组（或末端连接DSB修复）位点处观察到，以及这些聚合酶的RNA掺入对体外末端连接有什么影响。 这项工作将提供一个全面的了解如何聚合酶在特定的，并在一般情况下，结束加工因素，采用的DSB修复，这样的修复途径是准确的末端连接途径。 这将有助于解决这些加工因素的活动是如何控制的，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.