Gerodimos Christina A, Chang Howard H Y, Watanabe Go, Lieber Michael R
From the Departments of Pathology, Biochemistry & Molecular Biology, and Molecular Microbiology & Immunology and the Department of Biological Sciences, Section of Molecular & Computational Biology, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California 90033.
From the Departments of Pathology, Biochemistry & Molecular Biology, and Molecular Microbiology & Immunology and the Department of Biological Sciences, Section of Molecular & Computational Biology, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California 90033.
J Biol Chem. 2017 Aug 25;292(34):13914-13924. doi: 10.1074/jbc.M117.798850. Epub 2017 Jul 10.
In humans, nonhomologous DNA end-joining (NHEJ) is the major pathway by which DNA double-strand breaks are repaired. Recognition of each broken DNA end by the DNA repair protein Ku is the first step in NHEJ, followed by the iterative binding of nucleases, DNA polymerases, and the XRCC4-DNA ligase IV (X4-LIV) complex in an order influenced by the configuration of the two DNA ends at the break site. The endonuclease Artemis improves joining efficiency by functioning in a complex with DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) that carries out endonucleolytic cleavage of 5' and 3' overhangs. Previously, we observed that X4-LIV alone can stimulate Artemis activity on 3' overhangs, but this DNA-PKcs-independent endonuclease activity of Artemis awaited confirmation. Here, using nuclease and ligation assays, we find that stimulation of Artemis nuclease activity by X4-LIV and the efficiency of blunt-end ligation are determined by structural configurations at the DNA end. Specifically, X4-LIV stimulated Artemis to cut near the end of 3' overhangs without the involvement of other NHEJ proteins. Of note, this ligase complex is not able to stimulate Artemis activity at hairpins or at 5' overhangs. We also found that X4-LIV and DNA-PKcs interfere with one another with respect to stimulating Artemis activity at 3' overhangs, favoring the view that these NHEJ proteins are sequentially rather than concurrently recruited to DNA ends. These data suggest specific functional and positional relationships among these components that explain genetic and molecular features of NHEJ and V(D)J recombination within cells.
在人类中，非同源DNA末端连接（NHEJ）是修复DNA双链断裂的主要途径。DNA修复蛋白Ku对每个断裂的DNA末端的识别是NHEJ的第一步，随后核酸酶、DNA聚合酶和XRCC4-DNA连接酶IV（X4-LIV）复合物按受断裂位点处两个DNA末端构型影响的顺序进行迭代结合。核酸内切酶Artemis与DNA依赖性蛋白激酶催化亚基（DNA-PKcs）形成复合物发挥作用，DNA-PKcs对5'和3'突出端进行核酸内切酶切割，从而提高连接效率。此前，我们观察到单独的X4-LIV可以刺激Artemis对3'突出端的活性，但Artemis这种不依赖DNA-PKcs的核酸内切酶活性有待证实。在这里，我们使用核酸酶和连接测定法发现，X4-LIV对Artemis核酸酶活性的刺激以及平端连接的效率由DNA末端的结构构型决定。具体而言，X4-LIV刺激Artemis在3'突出端末端附近切割，而无需其他NHEJ蛋白的参与。值得注意的是，这种连接酶复合物无法刺激Artemis在发夹结构或5'突出端的活性。我们还发现，在刺激Artemis对3'突出端的活性方面，X4-LIV和DNA-PKcs相互干扰，这支持了这些NHEJ蛋白是按顺序而非同时被招募到DNA末端的观点。这些数据表明这些组分之间存在特定的功能和位置关系，这解释了细胞内NHEJ和V(D)J重组的遗传和分子特征。
