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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Requirements for DNA hairpin formation by RAG1/2
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Requirements for DNA hairpin formation by RAG1/2

机译：RAG1 / 2对DNA发夹形成的要求

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摘要

The rearrangement of antigen receptor genes is initiated by double-strand breaks catalyzed by the RAG1/2 complex at the junctions of recombination signal sequences and coding segments. As with some "cut-and-paste" transposases, such as Tn5 and Hermes, a DNA hairpin is formed at one end of the break via a nicked intermediate. By using abasic DNA substrates, we show that different base positions are important for the two steps of cleavage. Removal of one base in the coding flank enhances hairpin formation, bypassing a requirement for a paired complex of two signal sequences. Rescue by abasic substrates is consistent with a base-flip mechanism seen in the crystal structure of the Tn5 postcleavage complex and may mimic the DNA changes on paired complex formation. We have 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 the indole ring. A W956A mutation in RAG1 had an inhibitory effect on both nicking and hairpin stages that could be rescued by abasic substrates. W956 is therefore a likely candidate for interacting with this base during hairpin formation.
机译：抗原受体基因的重排是由重组信号序列和编码片段交界处的RAG1 / 2复合物催化的双链断裂引发的。与某些“剪切粘贴”转座酶（例如Tn5和Hermes）一样，在DNA的发夹处通过一个刻痕的中间体在断裂的一端形成。通过使用无碱基DNA底物，我们表明不同的碱基位置对于裂解的两个步骤很重要。去除编码侧翼中的一个碱基增强了发夹的形成，从而绕过了对两个信号序列的成对复合体的要求。无碱基底物的拯救与在Tn5后切割复合物的晶体结构中看到的碱基翻转机制一致，并且可以模拟成对复合物形成时的DNA变化。我们在RAG1中搜索了一个色氨酸残基，该残基与Tn5中W298的功能相当，该残基通过将翻转的碱基堆积在吲哚环上来稳定DNA相互作用。 RAG1中的W956A突变对缺刻和发夹阶​​段均具有抑制作用，可以通过无碱基底物来挽救它。因此，W956是发夹形成过程中与此碱基相互作用的可能候选者。

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