Characterization of V(D)J cleavage and repair complexes

描述(申请人提供)：脊椎动物淋巴细胞抗原受体谱系的多样性在很大程度上是通过称为V(D)J重组的过程实现的，该过程通过一系列特定部位的DNA重组事件从组成基因片段的阵列中组装抗原受体基因。V(D)J复合现在被理解为包括两个不同的阶段。在第一阶段，两个蛋白质，称为RAG-1和RAG-2，在与重排受体编码片段相邻的重组信号序列(RSS)处引入DNA双链断裂(DSB)。在第二阶段，RAG蛋白产生的DSB中间体通过非同源末端连接(NHEJ)途径修复。损害RAG蛋白或NHEJ因子活性的突变是人类和动物免疫缺陷疾病的一个子集的基础。另一方面，某些形式的白血病和淋巴瘤似乎是由异常的V(D)J重组引起的。要了解其病因涉及V(D)J重排过程的疾病的起源，必须在分子水平上了解支持V(D)J重排的两个阶段的蛋白质-DNA复合体。以前的工作主要集中在定义RAG-RSS裂解复合体上。在这里，包含DNA弯曲和/或NHEJ因子的RAG-RSS复合体，代表V(D)J重组的切割和连接相之间的界面，将被系统地组装，并在体外使用迁移率移动和凝胶内酶分析以及DNA足迹技术进行表征。为此，提出了以下三个具体目标：(I)确定促进RAG介导的V(D)J重组信号突触和切割所需的HMG-1(DNA弯曲因子)的决定因素；(Ii)确定体外RAG-1和RAG-2在调节RSS识别、切割和/或与DSB修复因子结合方面起什么作用；以及(Iii)通过迁移率改变分析确定DSB修复因子与RAG-RSS复合体(-/+HMG-1)的关联，并表征这些新的蛋白质-DNA复合体的组成、DNA相互作用和活性。

DESCRIPTION (provided by applicant): Diversity in the vertebrate lymphocyte antigen receptor repertoire is largely achieved by a process termed V(D)J recombination that assembles antigen receptor genes from arrays of component gene segments by a series of site-specific DNA recombination events. V(D)J recombination is now understood to involve two distinct phases. In the first phase, two proteins, called RAG-1 and RAG-2, introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) abutting receptor coding segments undergoing rearrangement. In the second phase, the DSB intermediates generated by the RAG proteins are repaired via a non-homologous end-joining (NHEJ) pathway. Mutations that impair the activity of RAG proteins or the NHEJ factors underlie a subset of immunodeficiency disorders in humans and animals. On the other hand, certain forms of leukemia and lymphoma appear to arise from aberrant V(D)J recombination. Insight into the origins of diseases whose etiology involves the V(D)J rearrangement process necessarily requires a molecular level understanding of the protein-DNA complexes that support both phases of V(D)J recombination. Previous efforts have primarily focused on defining RAG-RSS cleavage complexes. Here, RAG-RSS complexes incorporating DNA bending and/or NHEJ factors, representing the interface between the cleavage and joining phases of V(D)J recombination, will be systematically assembled and characterized in vitro using mobility shift and in-gel enzyme assays, as well as DNA footprinting techniques. Toward this end, the following three specific aims are proposed: (i) to identify determinants of HMG-1 (a DNA bending factor) required to promote RAGmediated synapsis and cleavage of V(D)J recombination signals; (ii) determine what role catalytically "dispensable" portions of RAG-1 and RAG-2 play in vitro in modulating RSS recognition, cleavage and/or association with DSB repair factors; and (iii) identify DSB repair factor association with RAG-RSS complexes (-/+ HMG-1) by mobility shift assay and characterize the composition, DNA interactions and activity of these novel protein-DNA complexes.