RAG-induced DNA damage: mechanisms and responses

描述(由申请人提供)：V(D)J重组事件如何参与某些白血病和淋巴瘤的致癌染色体易位？已经提出了许多建议。一些事件似乎涉及RAG产生的DSB与伙伴座位上其他机制造成的断裂。几年前，我们提出，修复DSB的修复途径选择错误可能会导致异常的连接事件。我们最近的数据显示，RAG1或RAG2中的特定突变取消了途径选择，也破坏了体外切割后复合体的稳定性。我们的初步数据表明，其中一些突变与RAG诱导的小鼠淋巴瘤致癌易位有关。这些相同的突变与体内淋巴细胞中选择性NHEJ的增加有关。总的来说，这些数据支持以下假设，我们将在拟议的实验中进行测试。假设1：“路径选择控制”在维持基因组稳定性方面起着关键作用，并通过RAG裂解后复合体维持在V(D)J重组中。RAG裂解后复合体稳定性的降低(由多种原因中的任何一种引起)允许编码和/或信号末端对不适当的连接途径的可用性增加，从而促进异常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作为突变修复途径出现。在V(D)J重组的背景下理解对途径选择的控制以及禁用这一调控机制的后果将阐明一个近30年来一直没有得到回答的问题：发生在发育中的淋巴细胞的致癌易位是如何发生的？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处理这一重要问题。在拟议的研究中，如果我们的RAG突变体解除了对途径选择的调控，使我们能够在一个完整的经典途径的背景下观察到频繁的致癌基因重排，我们将能够建立替代的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，通过某种机制施加了对路径选择的控制。 公共卫生相关性：尽管导致白血病和淋巴瘤恶变的确切步骤尚不清楚，但很大一部分似乎是由于导致B和T淋巴细胞抗原受体基因多样化的生理性DNA重排过程中的错误所致。我们最近产生了提前释放DNA末端的重组酶突变体。有趣的是，这些突变会导致小鼠发生淋巴瘤；拟议的实验将使用这些小鼠和上一次资助期产生的其他分子工具来探索重组酶-DNA末端复合体的破坏导致淋巴肿大的机制。

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.