Han Z, Johnston C, Reeves W H, Carter T, Wyche J H, Hendrickson E A
Department of Molecular Biology, Brown University, Providence, Rhode Island 02912, USA.
J Biol Chem. 1996 Jun 14;271(24):14098-104. doi: 10.1074/jbc.271.24.14098.
Three proteins known to play a critical role in mammalian DNA double-strand break repair and lymphoid V(D)J recombination are the autoantigens Ku86 and Ku70 and a 465-kDa serine/threonine protein kinase catalytic subunit (DNA-PKcs). These proteins physically associate to form a complex (DNA.PK) with DNA-dependent protein kinase activity. In this study, we demonstrate using electrophoretic mobility shift assays (EMSAs) that the nuclear DNA end-binding activity of Ku is altered in the human promyelocytic leukemic HL-60 cell line. Western blot and EMSA supershift analyses revealed that HL-60 cells expressed both full-length and variant Ku86 proteins. However, a combined EMSA and immunoanalysis revealed that the Ku heterodimers complexed with DNA in HL-60 cells contained only the variant Ku86 proteins. Finally, UV cross-linking experiments and DNA.PK assays demonstrated that the Ku complexes containing variant Ku86 had a greatly reduced ability to interact with DNA-PKcs and that consequently HL-60 cells had severely diminished DNA.K activity. These data provide important insights into the interaction between Ku and DNA-PKcs and into the role of DNA.PK in DNA double-strand break repair.
已知在哺乳动物DNA双链断裂修复和淋巴细胞V(D)J重组中起关键作用的三种蛋白质是自身抗原Ku86和Ku70以及一种465 kDa的丝氨酸/苏氨酸蛋白激酶催化亚基（DNA-PKcs）。这些蛋白质通过物理结合形成具有DNA依赖性蛋白激酶活性的复合物（DNA-PK）。在本研究中，我们使用电泳迁移率变动分析（EMSA）证明，在人早幼粒细胞白血病HL-60细胞系中，Ku的核DNA末端结合活性发生了改变。蛋白质印迹和EMSA超迁移分析表明，HL-60细胞同时表达全长和变体Ku86蛋白。然而，EMSA和免疫分析相结合显示，HL-60细胞中与DNA复合的Ku异二聚体仅包含变体Ku86蛋白。最后，紫外线交联实验和DNA-PK分析表明，含有变体Ku86的Ku复合物与DNA-PKcs相互作用的能力大大降低，因此HL-60细胞的DNA-K活性严重降低。这些数据为Ku与DNA-PKcs之间的相互作用以及DNA-PK在DNA双链断裂修复中的作用提供了重要见解。
