DNA-BINDING AND COMPLEX FORMATION BY RAG PROTEINS

描述（改编自研究者摘要）：脊椎动物 免疫应答在很大程度上依赖于大量抗原- 受体。这些分子中的许多多样性现在已经被理解 由一种称为V（D）J的位点特异性DNA重组过程产生 重组遗传的编码区在反应中被剪切和粘贴 组装成熟基因。 组合和机械 对产品的贡献创造了一定程度的多样性， 通过遗传预先形成的基因获得。 两种蛋白质RAG 1和RAG 2， 在反应中发挥重要作用。 在过去的两年里，这些 有两种蛋白质被证明可以在适当的位置切割靶DNA， 不含其他因子的体外反应位点。 详细 研究这两种蛋白质单独和与其他蛋白质的作用， 反应后期步骤中的蛋白质对于理解至关重要 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。 DNA结合试验 基于UV交联将用于鉴定DNA结合 两种蛋白质的结构域。 将根据以下内容确定接触部位： DNA和蛋白质。 DNA结合结构域的定义将 也可以更好地理解这两种蛋白质的结构， 以及蛋白质-蛋白质相互作用中 联系人. (2)展示蛋白质-蛋白质相互作用， 单独的RAG蛋白。 两种蛋白质都没有单独显示出生物化学活性， 并且这两种蛋白质一起能够切割底物DNA， 不仅在单一信号，但在一对协调一致的方式， 信号. 可能涉及蛋白质-蛋白质相互作用。（三）、 研究蛋白质和DNA的相互作用，在假定的更大的复杂。 突变细胞，在某些DNA修复蛋白缺陷，不能 完成V（D）J复合反应。 识别的蛋白质 此行为是后续步骤的潜在参与者， 复合反应 因为这个复合物的一个候选蛋白质 是一种激酶，特别注意磷酸化 可能伴随复杂形成的事件。 这种重组 反应是正常免疫系统发展的重要步骤， 系统 此外，这种反应中的错误也涉及到 造成了染色体易位， 儿童白血病 最后，这种代谢途径与 DNA断裂修复和细胞周期调控， 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.