ResUlt6 在体外BbRAG1L和bbRAG2L形成一个TIR特异性核酸内切酶

在上面的ReSulT中，我们介绍了BbRAG1L和bbRAG2L的特点和在在体转移（ex vivo）的情况，这次我们要研究在体外（in vitro）的情况。

在这次的实验中，首先

We co-purified full-length bbRAG1L and bbRAG2L, each fused at its N terminus to maltose binding protein (MBP), from mammalian cells and performed cleavage reactions using substrates containing either a 5′-TIR/3′-TIR pair or a 12RSS/23RSS pair

所谓TRI pair，RSS pair就是两个转座子对（Figure 5B）

反应需要镁离子和一种人DNA结合蛋白HMGB1。可以看到bbRAG1L/bbRAG2L选手非常给力

Co-expressed bbRAG1L/bbRAG2L exhibited robust cleavage activity on the TIR substrate, with cleavage products detectable as early as 2 min.

我们想对比RAG1/2和bbRAG1L/2L的功能，于是

co-expressed RAG1/2 cleaved the RSS substrate with similar kinetics (lanes 6–10). Cleavage products had the sizes expected for double-strand breaks at the borders of the TIRs or RSSs, with both enzymes generating substantial amounts of the double-cleavage product (double asterisks). Some differences were noted in the pattern of cleavage products generated by bbRAG1L/2L as compared to RAG1/2.

那么不同在哪里？

BbRAG1L/2L generated a product corresponding to single cleavage at the 3′ TIR (asterisk), which was visible at 2 min and accumulated to high levels over the 30-min time course. In contrast, RAG1/2 generated predominantly the double-cleavage product, with single-cleavage products visible as minor species only at later time points, consistent with RAG’s well-known propensity to perform coordinated cleavage at a 12/23 RSS pair

从图中也可以看出，RAG1/2主要产生两边的切断，而BbRAG1L/2L主要产生一边的切断。

之后，我们开始探究BbRAG1L/2L产生活性的条件，我们发现BbRAG1L/2L两种蛋白必须同时存在，并且突变掉D701之后，该酶也失活了。同时，该任性的酶在RSS位点活性非常弱。更甚于它的兄弟RAG1/2，BbRAG1L/2L更加依赖HMGB1以及Mg2+、Mn2+两种金属阳离子。

为了测试BbRAG1L/2L和RAG1/2是否都是相似的发夹剪切机制（图上有）

cleavage reactions were performed using an end-labeled 3′-TIR DNA substrate, with the cleavage products analyzed by denaturing gel electrophoresis

实验的结果证明：确实一样。