DNA-Binding and Complex Formation by RAG Proteins

脊椎动物免疫反应的决定性特征是抗体和t细胞受体分子库的建立。单独来说，每一种分子对一种抗原都是特异性的，但生物体产生的这些分子种类繁多，因此，总的来说，可以实现广泛的保护。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是至关重要的。现在人们了解到，大部分的多样性源于一种称为V(D)J重组的位点特异性DNA重组过程。前B细胞和前t细胞的生殖系中遗传的编码区在组装成熟基因的反应中被“剪切和粘贴”。组合连接的数学原理和由于连接机制的故意不精确而在产品中引入的额外变异创造了一定程度的多样性，这是通过遗传预先形成的基因无法获得的。RAG1和RAG2两种蛋白在该反应中起重要作用。在体外反应中，这两种蛋白质被证明可以在合适的位点切割目标DNA，称为重组信号序列（RSS）。V(D)J复合表示一台极为精密的机器的作用。我们发现RAG1和RAG2形成了一个没有DNA的四聚体，并可能在反应过程中形成更高阶的结构。在没有或存在DNA的情况下，以及在反应的后期步骤中与其他蛋白质一起，对RAG蛋白的性质和行为的详细研究对于理解这一关键机制至关重要。我们将利用化学交联、生化和分子生物学等方法揭示RAG蛋白在V(D)J重组各步骤中的功能。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.