Structural Electron Microscopy Group, Section of Structural Biology, The Institute of Cancer Research, London SW3 6JB, UK.
Nucleic Acids Res. 2011 Jul;39(13):5757-67. doi: 10.1093/nar/gkr146. Epub 2011 Mar 30.
The multi-subunit DNA-dependent protein kinase (DNA-PK), a crucial player in DNA repair by non-homologous end-joining in higher eukaryotes, consists of a catalytic subunit (DNA-PKcs) and the Ku heterodimer. Ku recruits DNA-PKcs to double-strand breaks, where DNA-PK assembles prior to DNA repair. The interaction of DNA-PK with DNA is regulated via autophosphorylation. Recent SAXS data addressed the conformational changes occurring in the purified catalytic subunit upon autophosphorylation. Here, we present the first structural analysis of the effects of autophosphorylation on the trimeric DNA-PK enzyme, performed by electron microscopy and single particle analysis. We observe a considerable degree of heterogeneity in the autophosphorylated material, which we resolved into subpopulations of intact complex, and separate DNA-PKcs and Ku, by using multivariate statistical analysis and multi-reference alignment on a partitioned particle image data set. The proportion of dimeric oligomers was reduced compared to non-phosphorylated complex, and those dimers remaining showed a substantial variation in mutual monomer orientation. Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break.
多亚基 DNA 依赖性蛋白激酶（DNA-PK）是高等真核生物中非同源末端连接修复的关键酶，由一个催化亚基（DNA-PKcs）和 Ku 异源二聚体组成。Ku 招募 DNA-PKcs 到双链断裂处，在那里 DNA-PK 组装在 DNA 修复之前。DNA-PK 与 DNA 的相互作用通过自身磷酸化来调节。最近的 SAXS 数据解决了在纯化的催化亚基中发生的自身磷酸化引起的构象变化。在这里，我们通过电子显微镜和单颗粒分析首次对自身磷酸化对三聚体 DNA-PK 酶的影响进行了结构分析。我们观察到自身磷酸化物质存在相当大的异质性，通过使用多变量统计分析和多参考对准在分区粒子图像数据集上，我们将其解析为完整复合物的亚群，以及单独的 DNA-PKcs 和 Ku。与非磷酸化复合物相比，二聚体寡聚物的比例降低，而剩余的二聚体在相互单体取向方面表现出很大的变化。总之，我们的数据表明，DNA-PK 全酶在自身磷酸化后发生了实质性的重塑，这对于从修复的 DNA 双链断裂释放蛋白因子至关重要。
