TdT and pol mu in DNA repair and immune system diversity

TdT 和 pol mu 在 DNA 修复和免疫系统多样性中的作用

• 批准号: 6919896
• 负责人: DALE A RAMSDEN
• 金额: $ 25.99万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6919896
• 关键词: CHO cells; DNA directed DNA polymerase; DNA repair; deoxyribonucleoside triphosphate; double stranded RNA; enzyme activity; gene induction /repression; genetic recombination; mass spectrometry; molecular assembly /self assembly; neoplasm /cancer immunology; polymerase chain reaction; protein kinase C; protein protein interaction; restriction fragment length polymorphism; terminal nick end labeling

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.

描述（由申请人提供）：在所有细胞类型中使用哺乳动物的非同源末端连接（NHEJ，END连接）途径来修复由DNA损伤剂引起的DNA双链断裂（DSB）（例如电离辐射，化学治疗药物）。 在V（d）J重组期间有效解析DSB中间体也是必不可少的，这是组装免疫系统抗原特异性受体所需的淋巴特异性过程。 末端连接的有缺陷导致放射敏感性，癌症的发生率增加以及免疫缺陷。 两种相关的DNA聚合酶，分别是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MU（POL MU），特别是与结束连接所需的因素相关的。 1）POL MU在结束时的作用尚不清楚。 细胞V（d）J重组测定法将用于阐明POL MU在V（d）J重组中的作用，及其在最终连接修复中的作用。 2）先前的细胞实验概述了末端连接的聚合酶的特征活动，因此该途径比其他预期的要准确。 使用纯化因子的减少系统将用于确定POL MU，TDT或其他聚合酶是否具有这些特征活动。 也将使POL MU或TDT中的目标突变更好地理解这些特定聚合酶与最终连接因子相关的能力的重要性，以实现更准确的结束连接。 3）TDT和POL MU在体外合成过程中很容易掺入DNA和RNA，而所有其他已知的核酸聚合酶（RNA或DNA）通常将适当的核酸类型掺入至少比不适当的核酸类型的效率高1000倍。 将进行实验确实确定是否还可以在细胞中V（d）J重组（或结束DSB修复）的位点观察到这种活性，以及​​这些聚合酶掺入的RNA掺入的影响对体外结合的最终连接。 这项工作将提供一个综合的理解，该聚合酶通常通过连接DSB维修途径的特定和最终处理因子中的聚合酶如何准确。 它将有助于解决这些处理因子的活性如何控制，如何以可接受的风险来实现结束处理，以及未控制的处理活动的后果可能是基因组稳定性的。