DNA-Binding and Complex Formation by RAG Proteins

脊椎动物免疫反应的决定性特征是建立了一系列抗体和T细胞受体分子。单独而言，每个分子都是针对单一抗原的，但一个生物体会产生种类繁多的这些分子，因此，总的来说，实现了广泛的保护。由于这些受体是由基因编码的，因此如何在遗传水平上获得这种多样性的问题至关重要。许多多样性现在被认为是来自一种称为V(D)J重组的特定部位的DNA重组过程。在组装成熟基因的反应中，前B和前T细胞胚系中遗传的编码区被“剪切和粘贴”。由于连接机制的刻意不精确，产品中引入了组合连接和附加变异的数学运算，创造了通过遗传预先形成的基因无法获得的多样性程度。RAG1和RAG2这两种蛋白质在该反应中起着至关重要的作用。这两种蛋白质已被证明在体外反应中在适当的位置切割目标DNA，称为重组信号序列(RSS)。V(D)J复合代表了一台精密机器的动作。我们发现RAG1和RAG2在反应过程中形成了一个没有DNA的四聚体，并可能形成更高的有序结构。详细研究RAG蛋白在没有或存在DNA的情况下以及在反应后期与其他蛋白质协同作用的性质和行为，对于理解这一关键机制是至关重要的。我们将使用化学交联、生化和分子生物学的方法来揭示RAG蛋白在V(D)J重组的每一步中的功能。了解这一机制将为DNA重组和其他高度有序的酶系统提供一个有价值的模型。

The defining feature of the vertebrate immune response is the establishment of a repertoire of antibody and T-cell receptor molecules. Individually, each molecule is specific for a single antigen, but an organism generates a tremendous variety of these molecules so that, in aggregate, a broad protection is achieved. Since these receptors are encoded by genes, the question of how such diversity is acquired at the genetic level is crucial. Much of the diversity is now understood to arise from a site-specific DNA recombination process called V(D)J recombination. Coding regions inherited in the germ line of pre- B and pre-T cells are "cut and pasted" in a reaction that assembles the mature gene. The mathematics of the combinatorial joining and additional variation introduced in the product by the deliberate imprecision of the joining mechanism 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. These two proteins have been shown to cut the target DNA at the appropriate site, called the Recombination Signal Sequence (RSS), in an in vitro reaction. V(D)J recombination represents the action of an exquisitely precise machine. We showed that RAG1 and RAG2 form a tetramer without DNA and may form even higher order structures during the reaction. A detailed study of the properties and behavior of the RAG proteins in the absence or presence of DNA and in concert with other proteins in the later steps of the reaction is vital to an understanding of this critical mechanism. We will use chemical crosslinking, biochemical and molecular biological methods to reveal the functions of the RAG proteins in each step in V(D)J recombination. Understanding this mechanism will provide a valuable model for DNA recomb ination and additional highly ordered enzyme systems.