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

参与 V(D)J 重组的蛋白质的结构研究

• 批准号: 10697747
• 负责人: MARTIN F. GELLERT
• 金额: $ 124.07万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697747
• 关键词: Adaptive Immune System; Binding; Biochemical; Chordata; Code; Collaborations; Complex; Cryoelectron Microscopy; DNA; DNA Binding; DNA-dependent protein kinase; Enzymes; Ephrin-A5; Eukaryota; Genes; Human; Immunoglobulins; Nonhomologous DNA End Joining; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Process; Proteins; Publishing; Shark; Site; Structure; T-Cell Receptor Genes; V(D)J Recombination; Work; Yang; artemis; endonuclease; experimental study; nuclease

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:

V(D)J 重组中一个长期存在的问题是，在连接完整的编码序列之前，需要打开 DNA 切割产生的 DNA 发夹。发夹只能由阿耳忒弥斯核酸内切酶切割，但该酶是自我抑制的，但其激活方式尚不清楚。在与杨伟博士的持续合作中，我们现在通过冷冻电镜和生化实验的结合证明，在 DNA-PK 在一组特定位点自动磷酸化后，Artemis 在与 DNA 依赖性蛋白激酶 (DNA-PK) 的复合物中变得活跃，从而导致其结构大规模打开，为 Artemis 提供了在 DNA 发夹附近结合的空间，并且 同时激活其核酸酶活性。 DNA-PK 由其自身磷酸化引起的结构变化也会抑制其激酶活性，因此其（必要的）参与非同源末端连接的后期阶段将需要进一步的处理，可能涉及磷酸酶。 有趣的一点是 DNA-PK 对结合 DNA 的结构敏感。对于非发夹末端，它更有可能避免自身磷酸化，而是转化为更有可能磷酸化其他蛋白质的状态。 该作品已发表：