Lehman Jason A, Hoelz Derek J, Turchi John J
Biomedical Sciences Graduate Program, Wright State University, Dayton, Ohio 45435, USA.
Biochemistry. 2008 Apr 15;47(15):4359-68. doi: 10.1021/bi702284c. Epub 2008 Mar 21.
Ionizing radiation induces DNA double-strand breaks which are repaired by the nonhomologous end joining (NHEJ) pathway. NHEJ is initiated upon Ku binding to the DNA ends and facilitating an interaction with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This heterotrimeric DNA-PK complex is then active as a serine/threonine protein kinase. The molecular mechanisms involved in DNA-PK activation are unknown. Considering the crucial role of Ku in this process, we therefore determined the influence of DNA binding on the structure of the Ku heterodimer. Chemical modification with NHS-biotin and mass spectrometry were used to identify sites of modification. Biotinylation of free Ku revealed several reactive lysines on Ku70 and Ku80 which were reduced or eliminated upon DNA binding. Interestingly, in the predicted C-terminal SAP domain of Ku70, biotinylation patterns were observed which suggest a structural change in this region of the protein induced by DNA binding. Limited proteolytic digests of free and DNA-bound Ku revealed a series of unique peptides, again, indicative of a change in the accessibility of the Ku70 and Ku80 C-terminal domains. A 10 kDa peptide was also identified which was preferentially generated under non-DNA-bound conditions and mapped to the C-terminus of Ku70. These results indicate a DNA-dependent movement or structural change in the C-terminal domains of Ku70 and Ku80 that may contribute to DNA-PKcs binding and activation. These results represent the first demonstration of DNA-induced changes in Ku structure and provide a framework for analysis of DNA-PKcs and the mechanism of DNA-PK activation.
电离辐射会诱导DNA双链断裂，这些断裂由非同源末端连接（NHEJ）途径修复。NHEJ在Ku与DNA末端结合并促进其与DNA依赖性蛋白激酶催化亚基（DNA-PKcs）相互作用时启动。然后，这种异源三聚体DNA-PK复合物作为丝氨酸/苏氨酸蛋白激酶发挥活性。DNA-PK激活所涉及的分子机制尚不清楚。鉴于Ku在此过程中的关键作用，我们因此确定了DNA结合对Ku异二聚体结构的影响。使用NHS-生物素进行化学修饰和质谱分析来鉴定修饰位点。游离Ku的生物素化揭示了Ku70和Ku80上的几个反应性赖氨酸，它们在DNA结合后减少或消除。有趣的是，在Ku70预测的C末端SAP结构域中，观察到生物素化模式，这表明DNA结合诱导了该蛋白质区域的结构变化。对游离和DNA结合的Ku进行有限的蛋白水解消化，揭示了一系列独特的肽段，同样表明Ku70和Ku80 C末端结构域的可及性发生了变化。还鉴定出一种10 kDa的肽段，它在非DNA结合条件下优先产生，并定位到Ku70的C末端。这些结果表明Ku70和Ku80的C末端结构域中存在DNA依赖性的移动或结构变化，这可能有助于DNA-PKcs的结合和激活。这些结果首次证明了DNA诱导的Ku结构变化，并为分析DNA-PKcs和DNA-PK激活机制提供了框架。
