Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31077, Cedex 4, France.
J Cell Biol. 2013 Jan 21;200(2):173-86. doi: 10.1083/jcb.201203128.
Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)-DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)-X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4-Cer-XLF complexes on DNA ends.
非同源末端连接是真核多细胞生物中主要的脱氧核糖核酸（DNA）双链断裂修复途径。为了启动修复，Ku 结合 DNA 末端并招募依赖于 DNA 的蛋白激酶（DNA-PK）催化亚基（DNA-PKcs）形成全酶。早期末端连接与激酶自身磷酸化有关。XRCC4（X4）-DNA 连接酶 IV（LIG4）复合物（X4LIG4）执行由 Cernunnos（Cer）-X4 样因子（XLF）促进的最终连接。在本文中，我们使用一种体外无细胞系统来重现末端连接和 DNA-PKcs 自身磷酸化，发现缺乏 LIG4 的人细胞提取物在这两种活性上都存在缺陷。在使用纯化成分的重建测定中，LIG4 还刺激了 DNA-PKcs 的自身磷酸化。此外，我们还发现 LIG4 缺陷细胞存在激酶自身磷酸化缺陷，该缺陷可通过过表达无催化活性的 LIG4 得到纠正。最后，我们的数据支持 Cer-XLF 对连接复合物在末端连接中这种意外的早期作用的贡献。我们提出，有效的末端连接是通过在 DNA 末端早期形成包含 DNA-PK 和 X4LIG4-Cer-XLF 复合物的超分子实体来实现的。
