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内稳态至关重要。DNA双链断裂（DSB）是基因组损伤的最具细胞毒性的形式之一。DSB可由于暴露于DNA损伤剂或在正常DNA代谢过程中而产生。此外，程序性DSB是通过高度调控的过程产生的，如淋巴特异性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，这可能导致细胞和生物体的严重有害结果。非同源末端连接（NHEJ）途径是哺乳动物细胞中主要的DNA DSB修复途径之一，并且是一般DSB修复以及V（D）J重组所必需的。许多染色体DSB，包括在V（D）J重组期间产生的那些，具有在连接之前需要修饰的末端结构。因此，在NHEJ期间发生的一个重要事件是DNA末端的加工以准备它们用于连接。Artemis DNA核酸酶与DNA-PKcs蛋白激酶复合，作为内切核酸酶起着关键作用，并且可以在单链至双链转变处切割DNA底物，如瓣、突出端、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Mre 11，其在Mre 11/Rad 50/Nbs 1（MRN）复合物的背景下起作用。一些证据表明，Artemis和Mre 11可能在DNA双链断裂修复过程中协同发挥作用。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Mre 11中的突变导致与基因组不稳定相关的人类免疫缺陷，在某些情况下，还导致癌症易感性。因此，目前建议的主要目标是更好地了解Artemis和Mre 11之间的分子和功能相互作用，这些相互作用涉及淋巴细胞发育和一般DSB修复背景下的DNA末端加工。为此，提出了三个具体目标。目的1的目的是阐明在V（D）J重组过程中Artemis和Mre 11 DNA核酸酶之间的功能相互作用，使用新的小鼠模型，携带条件，无效和亚型敲入等位基因。目的2是研究在缺陷V（D）J重组的背景下Mre 11在肿瘤发生中的作用。在目标3中，我们提出了实验来阐明一般DNA DSB修复过程中Artemis和Mre 11之间的功能相互作用。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，拟议的研究将提供一个更深入的了解致癌事件的产生，导致肿瘤发生的机制。

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.