Structural studies of proteins involved in V(D)J recombination

在V(D)J重组中一直存在的一个问题是，DNA切割产生的DNA发夹需要在完整的编码序列连接之前打开。发夹只能由Artemis核酸内切酶切割，但这种酶是自我抑制的，它是如何被激活的还不清楚。在我们与杨巍博士的持续合作中，通过低温电镜和生化实验的结合，我们现在已经证明，在DNA- pk在一组特定位点被自动磷酸化后，Artemis在与DNA依赖性蛋白激酶（DNA- pk）的复合物中变得活跃，这导致其结构的大规模开放，为Artemis提供了在DNA发卡附近结合的空间，同时激活其核酸酶活性。DNA-PK的自磷酸化引起的结构变化也抑制了它的激酶活性，因此它（必要的）参与非同源末端连接的后期阶段将需要进一步的加工，可能涉及一个磷酸酶。

One persistent question in V(D)J recombination has been that the DNA hairpins produced by DNA cleavage need to be opened before the complete coding sequence can be joined. Hairpins are cut only by the Artemis endonuclease, but that enzyme is self-inhibitedhow it is activated has been obscure. In our continuing collaboration with Dr. Wei Yang, we have now shown, by a combination of cryo-EM and biochemical experiments, that Artemis becomes active in complex with the DNA-dependent protein kinase (DNA-PK), after DNA-PK is auto-phosphorylated at a specific set of sites, which leads to a large-scale opening of its structure, giving Artemis a space to bind near the DNA hairpin and simultaneously activating its nuclease activity. The structural changes in DNA-PK caused by its auto-phosphorylation also inhibit its kinase activity, so that its (necessary) participation in the later stages of non-homologous end-joining will require further processing, likely to involve a phosphatase. An interesting point is that DNA-PK is sensitive to the structure of bound DNA. With non-hairpin ends, it is more likely to avoid auto-phosphorylation and instead convert to a state where it is more likely to phosphorylate other proteins. This work has been published: