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首页> 外文期刊>Molecular and Cellular Biology >Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination
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Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination

机译：位于V（D）J重组中顺式和DNA欠绕的重组信号序列的突触

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V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.
机译：V（D）J重组需要RAG1和RAG2蛋白在高迁移率基团蛋白HMG1或HMG2的辅助下结合和突触互补（12/23）对重组信号序列（RSS）。这种突触或成对复合物中的双链DNA裂解被认为与DNA的裂解或裂解位点附近的熔解有关。尽管V（D）J重组通常发生在位于同一DNA分子（在 cis 中）的RSS之间，但以前所有直接评估RSS突触的研究都是在 trans < / em>。为了克服这一局限性，我们开发了一种便利的环化分析方法，该方法使用长度减小的DNA底物来评估 cis 中RSS的突触。我们显示12/23对RSS是顺式 RSS的突触的首选底物，并且配对的效率取决于RAG1-RAG2的化学计量。 顺式中的突触迅速发生，并且在动力学上优于位于 trans 中的RSS的突触。该实验系统还允许生成包含顺式中的RSS对的未包裹DNA底物。重要的是，我们发现与松弛的底物相比，RAG蛋白可更有效地裂解此类底物，并且欠卷可增强RSS突触以及RAG1 / 2介导的催化作用。储存在这样的伤口基底中的能量可以用于产生突触和切割所需的DNA变形和/或蛋白质构象变化。我们建议这种放松是在适当的12/23对RSS的突触过程中唯一感受到的。

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