He D M, Lee S E, Hendrickson E A
Department of Molecular Biology, Cellular Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
Mutat Res. 1996 May 15;363(1):43-56. doi: 10.1016/0921-8777(95)00060-7.
Ku is a heterodimeric protein composed of 86 and 70 kDa subunits that binds preferentially to the double-stranded ends of DNA. Recent molecular characterization of ionizing-radiation sensitive (IRs) mutants belonging to the XRCC5 complementation group demonstrated the involvement of Ku in DNA double-strand break (DSB) repair and lymphoid V(D)J recombination. Here, we describe the isolation of a full-length hamster cDNA encoding the large subunit of the Ku heterodimer and demonstrate that the stable expression of this cDNA can functionally restore IR, Ku DNA end-binding activity and V(D)J recombination proficiency in the Chinese hamster IRs sxi-3 mutant. Moreover, we also demonstrate that sxi-3 cells are hypersensitive to etoposide, a DNA topoisomerase II inhibitor, and that resistance to this drug was restored by the Ku86 cDNA. These experiments suggest that a defect in the large subunit of the heterodimeric Ku protein is the sole factor responsible for the known defects of sxi-3 cells and our data of further support the role of Ku in DNA DSB repair and V(D)J recombination.
Ku是一种由86 kDa和70 kDa亚基组成的异源二聚体蛋白，它优先结合DNA的双链末端。最近对属于XRCC5互补组的电离辐射敏感（IRs）突变体进行的分子特征分析表明，Ku参与DNA双链断裂（DSB）修复和淋巴细胞V(D)J重组。在此，我们描述了编码Ku异源二聚体大亚基的全长仓鼠cDNA的分离，并证明该cDNA的稳定表达可在中华仓鼠IRs sxi-3突变体中功能性地恢复IR、Ku DNA末端结合活性和V(D)J重组能力。此外，我们还证明sxi-3细胞对DNA拓扑异构酶II抑制剂依托泊苷高度敏感，并且Ku86 cDNA可恢复对该药物的抗性。这些实验表明，异源二聚体Ku蛋白大亚基的缺陷是导致sxi-3细胞已知缺陷的唯一因素，我们的数据进一步支持了Ku在DNA DSB修复和V(D)J重组中的作用。
