DNA-BINDING AND COMPLEX FORMATION BY RAG PROTEINS

描述(摘自《调查者摘要》)：脊椎动物 免疫反应在很大程度上依赖于大量的抗原- 感受器。这些分子中的大部分多样性现在都被理解了 产生于一种称为V(D)J的位点特异性DNA重组过程 重组。遗传的编码区在反应中被剪切和粘贴 组装成熟的基因。组合论和机械论 对产品的贡献创造了一定程度的多样性，这是 是通过遗传预先形成的基因获得的。RAG1和RAG2两种蛋白质， 在反应中起着至关重要的作用。在过去两年中，这些 已经证明有两种蛋白质可以在适当的位置切割靶DNA 不含其他因素的体外反应中的结合部位。一个详细的 这两种蛋白质单独及与其他蛋白质协同作用的研究 蛋白质在反应的后期步骤对于理解 这一关键机制。具体目标是：(1)确定 单个RAG蛋白和DNA之间的接触。一种DNA结合试验 基于紫外光的交联会被用来鉴定DNA的结合 这两种蛋白质的结构域。联系地点将在以下日期确定 DNA和蛋白质上。DNA结合域的定义将 还可以更好地理解这两种蛋白质的结构， 以及蛋白质-蛋白质中应涉及的结构域的划分 联系人。(2)展示蛋白质与蛋白质之间的相互作用 仅RAG蛋白。这两种蛋白质都没有单独表现出生化活性， 这两种蛋白质结合在一起能够切割底物DNA， 不仅是在单个信号下，而且在成对的 信号。很可能涉及蛋白质之间的相互作用。(3) 研究假定更大的复合体中的蛋白质和DNA的相互作用。 突变细胞，在某些DNA修复蛋白上有缺陷，无法 完成V(D)J复合反应。被识别的蛋白质 此行为是 复合反应。由于该复合体的一种候选蛋白质 是一种激酶，将特别注意磷酸化。 可能伴随复杂队形的事件。这种重组 反应是形成正常免疫的必要步骤。 系统。此外，这种反应中的错误被认为与 造成的染色体易位似乎会导致许多 儿童白血病。最后，这条代谢途径与 DNA断裂修复和细胞周期调节，使研究 V(D)J重组具有更广泛的意义。

DESCRIPTION (Adapted from Investigator's abstract): The vertebrate immune response is largely dependent on a vast population of antigen- receptors. Much of the diversity in these molecules is now understood to arise from a site-specific DNA recombination process called V(D)J recombination. Inherited coding regions are cut and pasted in a reaction that assembles mature genes. The combinatorial and mechanistic contributions to the product create a degree of diversity that could not be obtained by inheriting preformed genes. Two proteins, RAG1 and RAG2, play an essential role in the reaction. In the past two years, these two proteins have been shown to cut the target DNA at the appropriate site in an in vitro reaction that contains no other factors. A detailed study of the role of these two proteins alone and in concert with other proteins in the later steps of the reaction is vital to an understanding of this critical mechanism. The specific aims are: (1) Determine the contacts between individual RAG proteins and DNA. A DNA binding assay based on UV crosslinking will be used to identify the DNA binding domains of the two proteins. The contact sites will be determined on the DNA and on the protein. Definition of the DNA binding domain will also allow better understanding of the architecture of the two proteins, and delineation of domains that should be involved in protein-protein contacts. (2) Demonstrate protein-protein interactions involving the RAG proteins alone. Neither protein shows biochemical activity alone, and the two proteins together are capable of cleaving the substrate DNA, not only at single signals, but in a concerted manner at pairs of signals. Protein-protein interactions are likely to be involved. (3) Study protein and DNA interactions in the putative larger complex. Mutant cells, defective in certain DNA repair proteins, are unable to complete the V(D)J recombination reaction. The proteins recognized by this behavior are potential participants in later steps in the recombination reaction. Since one candidate protein for this complex is a kinase, particular attention will be directed to phosphorylation events that may accompany complex formation. This recombination reaction is an essential step in the development of a normal immune system. Furthermore, errors in this reaction have been implicated in creating the chromosomal translocations that seem to precipitate many childhood leukemias. Finally, this metabolic pathway is connected to DNA break repair and cell cycle regulation which gives the study of V(D)J recombination a broader significance.