RAG-induced DNA damage: mechanisms and responses

描述（由申请人提供）：V(D)J重组事件如何参与一些白血病和淋巴瘤的致癌性染色体易位的产生？提出了许多建议。有些事件似乎涉及将rag生成的dsb连接到伙伴位点上由其他机制产生的中断。几年前，我们提出修复途径选择错误可能导致异常连接事件。我们最近的数据显示，RAG1或RAG2的特异性突变在体外破坏了途径选择，也破坏了裂解后复合体的稳定性。我们的初步数据表明，在小鼠淋巴瘤中，ragg诱导的一些突变致癌易位。这些相同的突变与体内淋巴细胞中选择性NHEJ的增加有关。总之，这些数据支持以下假设，我们将在拟议的实验中进行检验。假设1：“途径选择控制”在维持基因组稳定性中起关键作用，并在RAG裂解后复合体的V(D)J重组中得以维持。RAG切割后复合体的稳定性下降（由多种原因引起）使得编码和/或信号末端的可用性增加到不适当的连接途径，促进异常V(D)J重组产物的形成，包括致癌染色体易位。假设2：取消途径选择控制允许替代NHEJ作为致突变修复途径出现。在V(D)J重组的背景下，理解通路选择的控制和使这种调节机制失能的后果将阐明一个近30年来仍未得到解答的问题：在发育中的淋巴细胞中发生的致癌易位是如何产生的？我们从拟议的研究中获得的知识也可能帮助我们在其他情况下解决这个重要问题。如果在这些研究中，我们的RAG突变体解除了对通路选择的调控，使我们能够在完整的经典通路的背景下观察到频繁的致癌重排，我们将能够确定替代的NHEJ确实可以与经典的连接机制竞争。这一结果表明，在其他不涉及RAG蛋白的DSB修复情况下，途径选择的控制是通过某种机制强加的。

DESCRIPTION (provided by applicant): How are V(D)J recombination events involved in generating oncogenic chromosome translocations underlying some leukemias and lymphomas? Many proposals have been advanced. Some events appear to involve joining of RAG-generated DSBs to breaks made by other mechanisms at the partner loci. A few years ago, we proposed that errors the choice of repair pathway to heal the DSB could lead to aberrant joining events. Our recent data now reveal that specific mutations in either RAG1 or RAG2 abrogate pathway choice and also destabilize the post-cleavage complex in vitro. Our preliminary data implicate some of these mutations in RAG-induced oncogenic translocations in murine lymphomas. These same mutations are associated with increased alternative NHEJ in lymphocytes in vivo. Together, these data support the following hypotheses, which we will test in the proposed experiments. Hypothesis 1: "Pathway choice control" plays a critical role in maintaining genomic stability, and is maintained in V(D)J recombination by the RAG post-cleavage complex. Decreased stability of the RAG postcleavage complex (resulting from any of a number of causes) allows increased availability of the coding and/or signal ends to inappropriate joining pathways, facilitating formation of aberrant V(D)J recombination products, including oncogenic chromosome translocations. Hypothesis 2: Abrogating pathway choice control allows alternative NHEJ to emerge as a mutagenic repair pathway. Understanding control of pathway choice and the consequences of disabling this regulatory mechanism in the context of V(D)J recombination will illuminate a question that has remained unanswered for almost 30 years: how do the oncogenic translocations that occur in developing lymphocytes arise? The knowledge we gain from the proposed studies is also likely to help us approach this important question in other contexts. If, in the proposed studies, our RAG mutants which deregulate pathway choice allow us to observe frequent oncogenic rearrangements in the context of an intact classical pathway, we will be able to establish that alternative NHEJ can indeed compete with the classical joining mechanisms. This result would imply that control of pathway choice is imposed, by some mechanism, in other DSB repair situations that do not involve the RAG proteins. PUBLIC HEALTH RELEVANCE: Although the exact steps that lead to malignant transformation in leukemia and lymphoma remain unknown, a large fraction appear to result from mistakes in the physiologic DNA rearrangement process responsible for diversifying antigen receptor genes in B and T lymphocytes. We have recently generated recombinase mutants that prematurely release the DNA ends. Interestingly, these mutants cause lymphomas in mice; the proposed experiments will use these mice and other molecular tools generated during the last funding period to explore the mechanisms by which disruption of the recombinase-DNA end complex leads to lymphomagenesis.