DNA looping by Ku and the DNA-dependent protein kinase
DNA looping by Ku and the DNA-dependent protein kinase
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DOI：
10.1073/pnas.94.9.4267
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发表时间：
1997-04-29
影响因子：
11.1
通讯作者：
Chen, DJ
中科院分区：
文献类型：
--
作者：
Cary, RB;Peterson, SR;Chen, DJ
DNA依赖性蛋白激酶（DNA-PK）是DNA双链断裂（DSB）修复和免疫球蛋白基因重排所必需的，并可能在转录调节中发挥作用。DNA-PK全酶由三个多肽亚基组成：DNA结合Ku 70/86异二聚体和一个约460-kDa的催化亚基DNA-PK被认为是在DNA双链断裂处组装，并在双链断裂修复中发挥结构和信号转导作用。原子力显微镜（AFM）的最新进展已经导致能够产生天然蛋白质和蛋白质核酸复合物的高分辨率图像而无需染色或金属涂层的技术。原子力显微镜提供了一种快速、直接的方法来研究DNA修复和基因调控中蛋白质-核酸的相互作用。大量的DNA分子在Ku的存在下形成环。DNA成环似乎不依赖于序列，不受DNA-PKcs存在的影响。在DNA存在和不存在的情况下，Ku的凝胶过滤表明Ku不形成非特异性聚集体。我们的结论是，当结合到DNA，Ku是能够自我协会。这些发现表明，Ku在DNA双链断裂处的结合将导致Ku自缔合和断裂DNA链的物理束缚。
The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription, The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 heterodimer and an approximate to 460-kDa catalytic subunit (DNA-PKcs), DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and protein nucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation, Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs, Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.