Mechanisms underlying the generation of oncogenic chromosomal translocations

描述(由申请人提供)：保持基因组的完整性是确保细胞内环境稳定的关键。基因组损伤最具细胞毒性的形式之一是DNA双链断裂(DSB)。双链断裂可能是由于接触DNA损伤剂或在正常的DNA代谢过程中发生的。此外，程序性DSB是通过高度调控的过程产生的，例如淋巴特异性DNA重排、V(D)J重组。如果不修复或错误修复，双链断裂会导致基因组的不稳定性增加和染色体异常重排的积累，从而对细胞和生物体造成严重的有害后果。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，是一般DSB修复和V(D)J重组所必需的。许多染色体DSB，包括那些在V(D)J重组过程中产生的DSB，在连接之前都有需要修改的末端结构。因此，在NHEJ期间发生的一个重要事件是对DNA末端的处理，为连接做准备。Artemis DNA核酸酶与DNA-PKcs蛋白激酶形成复合体，作为核酸内切酶发挥重要作用，在单链到双链的转变过程中可以裂解DNA底物，如襟翼、悬垂、环和发夹。另一个在DSB修复过程中具有重要功能的核酸酶是Mre11，它在Mre11/Rad50/Nbs1(MRN)复合体中发挥作用。一些证据表明，Artemis和Mre11在DNA双链断裂修复过程中可能发挥协同作用。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，当前建议的主要目标是更好地了解Artemis和Mre11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修复过程中的DNA末端处理。为此，提出了三个具体目标。目标1的目的是利用含有条件性、零和亚型敲入等位基因的新型小鼠模型，阐明V(D)J重组过程中Artemis和Mre11DNA核酸酶之间的功能相互作用。目的2是在V(D)J重组缺陷的背景下，研究Mre11在肿瘤发生中的作用。在目标3中，我们提出了一些实验，以阐明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的染色体末端有效连接的分子事件提供重要的见解，这是维持基因组稳定和免疫系统发展所需的关键过程。此外，拟议的研究将提供更深入的了解潜在的致癌事件的产生，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.