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首页> 外文期刊>Molecular and Cellular Biology >Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences
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Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences

机译：RAG1和RAG2在绑定V（D）J重组信号序列中的不同作用

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The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three- to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1,10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border.
机译：RAG1和RAG2蛋白通过在重组信号序列（RSS）和编码段之间的边界处引入双链断裂来启动V（D）J重组。为了了解RAG1和RAG2在信号识别中的独特功能，我们通过凝胶阻滞和印迹分析比较了RAG1单独和RAG1加RAG2的DNA结合活性。与随机序列DNA相比，RAG1对于结合RSS的DNA仅表现出三到五倍的偏好。尽管未检测到RAG2本身的直接结合，但RAG1和RAG2的存在都会导致形成RAG1-RAG2-DNA复合物，该复合物比RAG1-DNA复合物更稳定，更特异性，并且在V（D）中具​​有活性J分裂这些结果表明，RSS和非特异性序列之间的生物学有效区分需要RAG1和RAG2。与RAG1和RAG2的结合不同，RAG1可以在不存在二价金属离子的情况下与DNA结合，并且不需要存在编码侧翼序列。具有1,10-菲咯啉-铜和硫酸二甲酯保护的RAG1-RAG2复合物的足迹表明，七聚体和九聚体均参与其中。九聚体受到保护，在小沟内与蛋白质广泛接触。相反，使七聚体更容易受到化学攻击，表明RAG1和RAG2的结合会扭曲编码/信号边界附近的DNA。

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