Mechanisms underlying the generation of oncogenic chromosomal translocations

描述（由申请人提供）：维持基因组的完整性对于确保细胞稳态至关重要。DNA双链断裂（DSB）是最具细胞毒性的基因组损伤形式之一。DSBs可由于暴露于DNA损伤剂或在正常的DNA代谢过程中产生。此外，程序化dsb是通过高度调控的过程产生的，如淋巴细胞特异性DNA重排，V(D)J重组。如果未修复或错误修复，dsb可能导致基因组不稳定性增加和异常染色体重排的积累，这可能导致对细胞和生物体严重有害的结果。非同源末端连接（non-homologous end joining， NHEJ）途径是哺乳动物细胞中DNA DSB修复的主要途径之一，是DSB修复和V(D)J重组所必需的。许多染色体dsb，包括在V(D)J重组过程中产生的，其末端结构需要在连接之前进行修饰。因此，在NHEJ过程中发生的一个重要事件是DNA末端的加工，为它们的结扎做准备。Artemis DNA核酸酶与DNA- pkcs蛋白激酶复合物，作为核酸内切酶起着关键作用，可以在单双链过渡时切割DNA底物，如皮瓣、悬垂、环和发夹。另一种在DSB修复过程中具有重要功能的核酸酶是Mre11，它在Mre11/Rad50/Nbs1 （MRN）复合体中起作用。一些证据表明，Artemis和Mre11可能在DNA dsb修复过程中协同起作用。然而，Artemis和MRN之间的确切作用和功能相互作用尚未阐明。Artemis或Mre11的突变会导致与基因组不稳定相关的人类免疫缺陷，在某些情况下还会导致癌症易感性。因此，本研究的主要目标是更好地了解Artemis和Mre11在淋巴细胞发育和DSB修复过程中参与DNA末端加工的分子和功能相互作用。为此，提出了三个具体目标。Aim 1的目的是阐明在V(D)J重组过程中Artemis和Mre11 DNA核酸酶之间的功能相互作用，使用新型小鼠模型，其中包含条件敲入等位基因、空敲入等位基因和半胚敲入等位基因。目的2是研究在V(D)J重组缺陷的情况下，Mre11在肿瘤发生中的作用。在Aim 3中，我们提出了实验来阐明Artemis和Mre11在一般DNA DSB修复过程中的功能相互作用。总之，这些研究将为确保断裂染色体末端有效连接的分子事件提供重要见解，这是维持基因组稳定性和免疫系统发育所需的重要过程。此外，拟议的研究将提供对导致肿瘤发生的致癌事件产生的机制的更深入的理解。

DESCRIPTION (provided by applicant): Maintaining the integrity of the genome is crucial in ensuring cellular homeostasis. One of the most cytotoxic forms of genomic damage is the DNA double strand break (DSB). DSBs can arise as a result of exposure to DNA damaging agents or during normal DNA metabolic processes. In addition, programmed DSBs are generated via highly regulated processes such as the lymphoid specific DNA rearrangement, V(D)J recombination. If unrepaired or misrepaired, DSBs can lead to increased genome instability and accumulation of aberrant chromosomal rearrangements which can result in severely detrimental outcomes for cells and organisms. The non-homologous end joining (NHEJ) pathway is one of the major DNA DSB repair pathways in mammalian cells and is required for general DSB repair as well as V(D)J recombination. Many chromosomal DSBs, including those generated during V(D)J recombination, have end structures that require modification prior to joining. Thus, one important event that occurs during NHEJ is the processing of the DNA ends to prepare them for ligation. The Artemis DNA nuclease, in complex with the DNA-PKcs protein kinase, plays a critical role as an endonuclease and can cleave DNA substrates at single to double strand transitions, such as flaps, overhangs, loops and hairpins. Another nuclease that has important functions during DSB repair is Mre11 which functions in the context of the Mre11/Rad50/Nbs1 (MRN) complex. Several lines of evidence suggest that Artemis and Mre11 may function in concert during the repair of DNA DSBs. However, the precise roles and functional interactions between Artemis and MRN have not been elucidated. Mutations in either Artemis or Mre11 result in human immunodeficiencies associated with genome instability, and in some cases, cancer predisposition. Thus, the major goals of the current proposal are to gain a better understanding of molecular and functional interactions between Artemis and Mre11 that are involved in DNA end processing in the context of lymphocyte development and general DSB repair. To this end, three specific aims are proposed. The goals of Aim 1 are to elucidate the functional interactions between the Artemis and Mre11 DNA nucleases during V(D)J recombination using novel mouse models harboring conditional, null, and hypomorphic knock-in alleles. Aim 2 Is to examine the roles of Mre11 in tumorigenesis in the context of defective V(D)J recombination. In Aim 3, we propose experiments to elucidate the functional interactions between Artemis and Mre11 during general DNA DSB repair. Together, these studies will provide important insights into the molecular events that ensure the efficient joining of broken chromosomal ends, a vital process required for maintenance of genome stability and immune system development. Furthermore, the proposed studies will provide a more in-depth understanding of the mechanisms underlying the generation of oncogenic events that lead to tumorigenesis. PUBLIC HEALTH RELEVANCE: This proposal will examine the molecular mechanisms underlying the repair of broken chromosomal ends and determine the consequences of aberrant DNA end processing on immune system development and lymphoid malignancies. Together, these studies will not only provide important insights into the basic processes required for maintenance of genome stability, but also lead to a better understanding of the causes of human immunodeficiency and cancer predisposition syndromes.