RAG-induced DNA damage: mechanisms and responses

描述（由申请人提供）：我们对预防染色体易位的安全机制感兴趣，染色体易位导致淋巴瘤和白血病的产生，这是最常见的人类癌症。每天有数百万淋巴细胞经历V（D）J重组，在个体的一生中，基因组完整性有许多机会被破坏-染色体易位实际上是淋巴肿瘤的主要特征。这些重排通常将完整的原癌基因置于高度表达的IG或TCR基因的调控下，导致对细胞生长、分化或凋亡的有害影响。令人信服的间接证据表明，V（D）J重组酶参与了这些易位，但它实际上如何参与异常重排仍然是个谜。罗斯实验室最近有几项发现，提出了V（D）J重组引发的异常重排的新机制。我们还发现了一个新的反应控制点，可以防止这些事件。这里提出的研究的首要目标是了解保持淋巴细胞基因组稳定性的机制，并确定V（D）J重组相关致癌重排的分子发病机制。为了这样做，我们将1）探测切割后复合物的支架作用的性质; 2）检查替代RAG切割机制对异常重排的贡献; 3）产生两个携带RAG突变的敲入小鼠系，我们预测该突变将增加异常重排的可能性; 4）描述DNA损伤传感器、检查点功能和V（D）J重组酶对维持基因组稳定性的具体贡献。最后一个目标是与Petrini实验室合作，并利用他们最近的工作，涉及Mrel I复合物控制V（D）J重组的保真度。

DESCRIPTION (provided by applicant): We are interested in the safety mechanisms that prevent chromosome translocations that lead to the generation of lymphomas and leukemias, which are among the most common human cancers. With millions of lymphocytes undergoing V(D)J recombination each day, there are many opportunities for genomic integrity to be breached over the course of an individual's lifetime-- and chromosomal translocations are, in fact, a cardinal feature of lymphoid neoplasms. These rearrangements typically place an intact proto-oncogene under the regulatory control of the highly expressed Ig or TCR genes, leading to deleterious effects on cell growth, differentiation, or apoptosis. Compelling circumstantial evidence has implicated the V(D)J recombinase in these translocations, but how it might actually participate in aberrant rearrangements has remained mysterious. The Roth laboratory has recently made several discoveries that suggest novel mechanisms for V(D)J recombination-initiated aberrant rearrangements. We have also uncovered a new control point in the reaction that may prevent these events. The overarching goal of the studies proposed here is to understand the mechanisms that preserve genomic stability in lymphocytes and to define the molecular pathogenesis of V(D)J recombination-associated oncogenic rearrangements. To do so we will 1) probe the nature of the post-cleavage complex's scaffolding role; 2) examine the contribution of alternative RAG cleavage mechanisms to aberrant rearrangements; 3) create two lines of knock-in mice bearing RAG mutations we predict will increase the likelihood of aberrant rearrangements; 4) delineate the specific contributions of DNA damage sensors, checkpoint functions and the V(D)J recombinase to maintaining genomic stability. The last aim is in collaboration with the Petrini lab and capitalizes on their recent work implicating the Mrel I complex in controlling the fidelity of V(D)J recombination.