The discovery of ProtoRAG in amphioxus indicated that vertebrate RAG recombinases originated from an ancient transposon. However, the sequences of ProtoRAG TIRs were obviously dissimilar to the consensus sequence of mouse 12/23RSS, and recombination mediated by ProtoRAG or RAG made them incompatible with each other. Thus, it is difficult to determine whether or how 12/23RSS persisted in the vertebrate RAG system that evolved from the TIRs of ancient RAG transposons. Here, we found that the activity of ProtoRAG is highly dependent on its asymmetric 5’TIR and 3’TIR, which are composed of conserved TR1 and TR5 elements and a partially conserved TRsp element of 27/31 bp to separate them. Similar to the requirements for the RSSs of RAG recombinase, the first CAC in TR1, the three dinucleotides in TR5 and the specific length of the partially conserved TRsp are important for the efficient recombination activity of ProtoRAG. In addition, the homologous sequences flanking the signal sequences facilitate ProtoRAG- but not RAG-mediated recombination. In addition to the diverged TIRs, two differentiated functional domains in BbRAG1L were defined to coordinate with the divergence between TIRs and RSSs. One of these is the CTT* domain, which facilitates the specific TIR recognition of the BbRAGL complex, and the other is NBD*, which is responsible for DNA binding and the protein stabilization of the BbRAGL complex. Thus, our findings reveal that the functional requirement for ProtoRAG TIRs is similar to that for RSS in RAG-mediated recombination, which not only supports the common origin of ProtoRAG TIRs and RSSs from the asymmetric TIRs of ancient RAG transposons but also reveals the development of RAG and RAG-like machineries during chordate evolution.

中文翻译：

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高效 ProtoRAG 活性的末端反向重复的功能要求揭示了 V(D)J 重组的早期进化

在两栖类动物中发现 ProtoRAG 表明脊椎动物 RAG 重组酶起源于一个古老的转座子。然而，ProtoRAG TIRs的序列与小鼠12/23RSS的共有序列明显不同，ProtoRAG或RAG介导的重组使它们彼此不相容。因此，很难确定 12/23RSS 是否或如何在从古代 RAG 转座子的 TIR 进化而来的脊椎动物 RAG 系统中持续存在。在这里，我们发现 ProtoRAG 的活性高度依赖于其不对称的 5'TIR 和 3'TIR，它们由保守的 TR1 和 TR5 元件和部分保守的 27/31 bp 的 TRsp 元件组成，以将它们分开。类似于对 RAG 重组酶 RSS 的要求，TR1 中的第一个 CAC，TR5 中的三个二核苷酸和部分保守的 TRsp 的特定长度对于 ProtoRAG 的有效重组活性很重要。此外，信号序列侧翼的同源序列促进 ProtoRAG 介导的重组，但不促进 RAG 介导的重组。除了分歧的 TIR 之外，还定义了 BbRAG1L 中的两个不同的功能域，以协调 TIR 和 RSS 之间的分歧。其中之一是 CTT* 结构域，它促进 BbRAGL 复合物的特异性 TIR 识别，另一个是 NBD*，它负责 DNA 结合和 BbRAGL 复合物的蛋白质稳定。因此，我们的研究结果表明，在 RAG 介导的重组中，ProtoRAG TIR 的功能要求与 RSS 的功能要求相似，