RAG-induced DNA damage: mechanisms and responses

描述(申请人提供)：我们对防止导致淋巴瘤和白血病产生的染色体易位的安全机制感兴趣，这是人类最常见的癌症之一。由于每天有数以百万计的淋巴细胞进行V(D)J重组，在人的一生中有许多机会破坏基因组的完整性--事实上，染色体易位是淋巴肿瘤的一个主要特征。这些重排通常将完整的原癌基因置于高表达的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重组酶与这些易位有关，但它实际上如何参与异常重排仍然是个谜。罗斯实验室最近的几项发现表明了V(D)J重组引发的异常重排的新机制。我们还发现了可能防止这些事件发生的反应中的一个新的控制点。这些研究的首要目标是了解维持淋巴细胞基因组稳定性的机制，并确定V(D)J重组相关的致癌基因重排的分子发病机制。为此，我们将1)探讨切割后复合体的支架作用的性质；2)检测替代的RAG切割机制对异常重排的贡献；3)创建两个携带RAG突变的敲入小鼠系，我们预测它们将增加异常重排的可能性；4)勾勒出DNA损伤感应器、检查点功能和V(D)J重组酶在维持基因组稳定性方面的具体贡献。最后一个目标是与Petrini实验室合作，并利用他们最近的工作，即MREL I复合体在控制V(D)J重组的保真度方面的作用。

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.