RAG-induced DNA damage: mechanisms and responses

RAG 诱导的 DNA 损伤：机制和反应

• 批准号: 7883901
• 负责人: DAVID B. ROTH
• 金额: $ 29.92万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7883901
• 关键词: Acidic Region; Address; Affect; Amino Acids; Antigen Receptors; Biological Assay; C-terminal; Cells; Choices and Control; Chromosomal translocation; Code; Complex; Cultured Cells; DNA; DNA Damage; DNA Sequence Rearrangement; Data; Double Strand Break Repair; Equation; Event; Funding; Gene Rearrangement; Genome; Genome Stability; Genomic Instability; Healed; Impairment; In Vitro; Knock-in Mouse; Knowledge; Lead; Libraries; Link; Lymphocyte; Lymphoma; Lymphomagenesis; Malignant - descriptor; Measures; Mediating; Molecular; Molecular Probes; Mus; Mutant Strains Mice; Mutate; Mutation; NBS1 gene; Nonhomologous DNA End Joining; Oncogenic; Pathway interactions; Physiological; Play; Process; Proteins; Receptor Gene; Regulatory Element; Role; Side; Signal Transduction; Small Interfering RNA; T-Lymphocyte; Tail; Testing; Transfection; V(D)J Recombination; Work; cell type; endodeoxyribonuclease SceI; healing; in vivo; interest; leukemia/lymphoma; mutant; novel; nuclease; prevent; protein complex; public health relevance; recombinase; repaired; research study; response; tool

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.

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的产生？已经提出了许多建议。一些事件似乎涉及 RAG 生成的 DSB 与伙伴基因座上其他机制造成的断裂的连接。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。我们最近的数据表明，RAG1 或 RAG2 中的特定突变会取消途径选择，并且还会破坏体外裂解后复合物的稳定性。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替代 NHEJ 的增加有关。这些数据共同支持以下假设，我们将在拟议的实验中对其进行测试。假设1：“路径选择控制”在维持基因组稳定性中发挥着关键作用，并通过RAG 裂解后复合物在V(D)J 重组中维持。 RAG 裂解后复合物的稳定性降低（由多种原因中的任何一种引起）会增加编码和/或信号末端对不适当连接途径的可用性，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：废除途径选择控制使得替代 NHEJ 作为诱变修复途径出现。了解通路选择的控制以及在 V(D)J 重组背景下禁用这种调节机制的后果将阐明一个近 30 年来仍未得到解答的问题：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。如果在拟议的研究中，我们的 RAG 突变体解除了途径选择的管制，使我们能够在完整的经典途径的背景下观察到频繁的致癌重排，那么我们将能够确定替代 NHEJ 确实可以与经典连接机制竞争。这一结果意味着在不涉及 RAG 蛋白的其他 DSB 修复情况下，通过某种机制强加了对途径选择的控制。 公共健康相关性：尽管导致白血病和淋巴瘤恶性转化的确切步骤仍不清楚，但很大一部分似乎是由于 B 和 T 淋巴细胞中抗原受体基因多样化的生理 DNA 重排过程中的错误造成的。我们最近产生了过早释放 DNA 末端的重组酶突变体。有趣的是，这些突变体会导致小鼠患淋巴瘤。拟议的实验将使用这些小鼠和上一个资助期间产生的其他分子工具来探索重组酶-DNA末端复合物的破坏导致淋巴瘤发生的机制。