Requirements for DNA hairpin formation in V(D)J recombination

与一些剪切粘贴转座一样，如Tn5和Hermes， RAG蛋白在DNA断裂的一端通过缺口中间物形成DNA发夹。利用基本的DNA底物，我们已经证明了不同的碱基位置对于切割的两个步骤是重要的，一些碱基对于切口是必不可少的，而另一些碱基则参与了发夹的形成。去除编码侧翼的一个特殊碱基，大大增强了发夹的形成，绕过了对两个重组位点的配对复合体的正常要求。这种基本底物的激活与一种机制是一致的，在这种机制中，一个碱基从双螺旋结构中翻转出来，并通过蛋白质相互作用稳定下来，从而允许发夹所需的尖锐DNA弯曲。这种碱基翻转在Tn5解理后络合物的晶体结构中可见；在RAG体系中，通常需要成对的复合体形成。我们还在RAG1中寻找了一个色氨酸残基，它的功能相当于Tn5中的W298，它通过将翻转的碱基堆叠在其吲哚环上来稳定DNA相互作用。我们确定RAG1的W956是最必需的色氨酸。该位点的突变极大地抑制了发夹反应，但正确的碱性底物可以挽救该反应。W956靠近RAG1中催化三联体酸性残基之一，因此可能在发夹形成过程中与翻转碱基相互作用。

As is true with some cut and paste transposases such as Tn5 and Hermes, the RAG proteins form a DNA hairpin at one end of the DNA break, via a nicked intermediate. Using abasic DNA substrates, we have shown that different base positions are important for the two steps of cleavage, some bases being essential for nicking while others are involved in hairpin formation. Removal of one particular base in the coding flank greatly enhances hairpin formation, bypassing the normal requirement for a paired complex of two recombination sites. Activation by this abasic substrate is consistent with a mechanism in which one base is flipped out of the double helix and stabilized by protein interactions, thus allowing the sharp DNA bend required for hairpinning. Such a base flip is seen in the crystal structure of the Tn5 post-cleavage complex; in the RAG system it may normally require paired complex formation. We have also searched for a tryptophan residue in RAG1 that would be the functional equivalent of W298 in Tn5, which stabilizes the DNA interaction by stacking the flipped base on its indole ring. We identified W956 of RAG1 as the most essential tryptophan. Mutations at this site greatly inhibited hairpinning, but the reaction could be rescued by the correct abasic substrate. W956, which is near one of the catalytic triad of acidic residues in RAG1, is therefore a likely candidate for interacting with the flipped base during hairpin formation.