An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex.
An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex.
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DOI：
10.1074/jbc.m116.751867
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发表时间：
2016-12-30
期刊：
影响因子：
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通讯作者：
Tainer JA
中科院分区：
文献类型：
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作者：
Hammel M;Yu Y;Radhakrishnan SK;Chokshi C;Tsai MS;Matsumoto Y;Kuzdovich M;Remesh SG;Fang S;Tomkinson AE;Lees-Miller SP;Tainer JA
人细胞非同源末端连接(NHEJ)的DNA双链断裂(DSB)修复是由Ku异源二聚体与DSB结合启动的，随后招募核心NHEJ因子，包括DNA依赖蛋白激酶催化亚单位(DNA-PKcs)、XRCC4样因子(XLf)和XRCC4(X4)-DNA连接酶IV(L4)。Ku还与辅助因子相互作用，如APRATAIN和多核苷酸激酶/磷酸酶样因子(APLF)。然而，这些因素如何相互作用来拴住、加工和连接DSB末端，同时允许调节和染色质相互作用，仍然是个谜。在这里，小角X射线散射(SAXS)和突变分析表明，APLF主要是一种本质上无序的蛋白质，它与Ku、Ku/DNA-PKcs(DNA-PK)和X4L4结合在一个扩展的柔性NHEJ核心复合体中。X4L4通过灵活的Ku80C-末端区域(Ku80CTR)与Ku、DNA-PK和X4L4通过APLF相互作用稳定的复合体与DNA-PKcs连接。集体结果揭示了六蛋白复合体的解决方案架构，并表明合作组装了一个扩展的灵活的NHEJ核心复合体，该复合体支持APLF的可及性，同时可能提供核心复合体与染色质的灵活连接。此外，由此产生的动态拴系提供了L4催化结构域在连接过程中到达DNA末端的几何通道，以及DNA-PKcs的几何通道，用于其他NHEJ蛋白的靶向磷酸化，以及DNA-PKcs在相对DSB上的反式磷酸化，而不破坏核心连接复合体。总体而言，这些结果阐明了Ku、X4和L4活性的进化保守，同时解释了Ku80CTR和DNA-PKcs只出现在高等真核生物的一个子集中的观察结果。
DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.