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首页> 外文期刊>Molecular and Cellular Biology >RAG-1 and RAG-2-dependent assembly of functional complexes with V(D)J recombination substrates in solution.
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RAG-1 and RAG-2-dependent assembly of functional complexes with V(D)J recombination substrates in solution.

机译：溶液中具有V（D）J重组底物的功能复合物的RAG-1和RAG-2依赖性组装。

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摘要

V(D)J recombination is initiated by RAG-1 and RAG-2, which introduce double-strand DNA breaks at recombination signal sequences (RSSs) of antigen receptor gene segments to produce signal ends, terminating in blunt, double-strand breaks, and coding ends, terminating in DNA hairpins. While the formation of RAG-RSS complexes has been documented, observations regarding the individual contributions of RAG-1 and RAG-2 to RSS recognition are in conflict. Here we describe an assay for formation and maintenance of functional RAG-RSS complexes in the course of the DNA cleavage reaction. Under conditions of in vitro cleavage, the RAG proteins sequester intact substrate DNA in a stable complex which is formed prior to strand scission. The cleavage reaction subsequently proceeds through nicking and hairpin formation without dissociation of substrate. Notably, the presence of both RAG-1 and RAG-2 is essential for formation of stable, functional complexes with substrate DNA under conditions of the sequestration assay. Two classes of substrate mutation are distinguished by their effects on RAG-mediated DNA cleavage in vitro. A mutation of the first class, residing within the RSS nonamer and associated with coordinate impairment of nicking and hairpin formation, greatly reduces the stability of RAG association with intact substrate DNA. In contrast, a mutation of the second class, lying within the RSS heptamer and associated with selective abolition of hairpin formation, has little or no effect on the half-life of the RAG-substrate complex.
机译：V（D）J重组是由RAG-1和RAG-2启动的，它们在抗原受体基因区段的重组信号序列（RSS）处引入双链DNA断裂以产生信号末端，并终止于平末端双链断裂，和编码末端，终止于DNA发夹。尽管已记录了RAG-RSS复合物的形成，但有关RAG-1和RAG-2对RSS识别的个体贡献的观察结果却相矛盾。在这里，我们描述了在DNA裂解反应过程中形成和维持功能性RAG-RSS复合物的方法。在体外切割的条件下，RAG蛋白将完整的底物DNA螯合在稳定的复合物中，该复合物是在断裂之前形成的。切割反应随后通过切口和发夹的形成而进行，而没有底物的解离。值得注意的是，RAG-1和RAG-2的存在对于在螯合测定条件下与底物DNA形成稳定的功能性复合物至关重要。两类底物突变的特征在于它们对体外RAG介导的DNA切割的影响。第一类突变位于RSS九聚体中，与切口和发夹形成的协调损伤相关，大大降低了RAG与完整底物DNA缔合的稳定性。相反，位于RSS七聚体中并与选择性消除发夹形成相关的第二类突变对RAG-底物复合物的半衰期影响很小或没有影响。

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