Structural studies of proteins involved in DNA repair and recombination

继续我们的工作，启动V（D）J重组的RAG1/2蛋白复合物，我们已经获得了一个更广泛的蛋白质结构，除了RAG1和RAG2的催化活性部分RAG2的完整的C-末端。这个C-末端延伸包含一个PHD结构域，该结构域结合到一个特定的组蛋白修饰：通过赖氨酸4上的三甲基化修饰的组蛋白3（称为H3K4Me），其表征活性染色质。因此，PHD结构域有助于将RAG1/2带到其所需的作用位点。此外，PHD结构域在不含组蛋白时抑制RAG1/2切割DNA。因此，PHD结构域在调节V（D）J重组中具有双重作用。我们的新结构清楚地说明了这种抑制是如何发生的。 继续我们对DNA依赖性蛋白激酶（DNA-PK）的研究，我们现在正在研究它在启动同源重组中的作用。已知这涉及其与Mre 11、Rad50和Nbs 1（MRN）的三蛋白复合物的相互作用，所述三蛋白复合物在DNA-PK封闭的DNA末端附近具有内切核酸酶和外切核酸酶活性，从而在DNA上产生必需的3突出端。这种具有挑战性的多蛋白质复合物（共七种蛋白质，包括辅因子）正在通过冷冻电子显微镜进行研究。

Continuing our work on the initiation of V(D)J recombination by the RAG1/2 protein complex, we have obtained a more extended protein structure containing the full C-terminal of RAG2 in addition to the catalytically active parts of RAG1 and RAG2 previously reported. This C-terminal extension contains a PHD domain that binds to a particular histone modification: histone 3 modified by trimethylation on Lysine 4 (known as H3K4Me) that characterizes active chromatin. The PHD domain thus helps to bring RAG1/2 to its desired sites of action. In addition, the PHD domain, when free of histone, inhibits RAG1/2 cleavage of DNA. Thus, the PHD domain pays a dual role in regulating V(D)J recombination. Our new structure makes it clear how this inhibition occurs. Continuing our work on the DNA-dependent protein kinase (DNA-PK), we are now looking at its role in initiating homologous recombination. This is known to involve its interaction with the three-protein complex of Mre11, Rad50, and Nbs 1 (MRN), which has both endo- and exonuclease activity near a DNA-PK blocked DNA end, thus creating the necessary 3 overhang on DNA. This challenging multi-protein complex (seven proteins altogether, including cofactors) is being studied by cryo-electron microscopy.