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

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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: