Jin S, Weaver D T
Division of Tumor Immunology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
EMBO J. 1997 Nov 17;16(22):6874-85. doi: 10.1093/emboj/16.22.6874.
Heterodimers of the 70 and 80 kDa Ku autoantigens (Ku70 and Ku80) activate the DNA-dependent protein kinase (DNA-PK). Mutations in any of the three subunits of this protein kinase (Ku70, Ku80 and DNA-PKcs) lead to sensitivity to ionizing radiation (IR) and to DNA double-strand breaks, and V(D)J recombination product formation defects. Here we show that the IR repair, DNA end binding and DNA-PK defects in Ku70-/- embryonic stem cells can be counteracted by introducing epitope-tagged wild-type Ku70 cDNA. Truncations and chimeras of Ku70 were used to identify the regions necessary for DNA end binding and IR repair. Site-specific mutational analysis revealed a core region of Ku70 responsible for DNA end binding and heterodimerization. The propensity for Ku70 to associate with Ku80 and to bind DNA correlates with the ability to activate DNA-PK, although two mutants showed that the roles of Ku70 in DNA-PK activation and IR repair are separate. Mutation of DNA-PK autophosphorylation sites and other structural motifs in Ku70 showed that these sites are not necessary for IR repair in vivo. These studies reveal Ku70 features required for double-strand break repair.
70 kDa和80 kDa Ku自身抗原（Ku70和Ku80）的异二聚体可激活DNA依赖性蛋白激酶（DNA-PK）。该蛋白激酶的三个亚基（Ku70、Ku80和DNA-PKcs）中任何一个发生突变都会导致对电离辐射（IR）敏感、DNA双链断裂以及V（D）J重组产物形成缺陷。在此我们表明，通过引入表位标签野生型Ku70 cDNA，可以抵消Ku70基因敲除胚胎干细胞中的IR修复、DNA末端结合和DNA-PK缺陷。利用Ku70的截短体和嵌合体来鉴定DNA末端结合和IR修复所需的区域。位点特异性突变分析揭示了Ku70中负责DNA末端结合和异二聚化的核心区域。Ku70与Ku80结合以及与DNA结合的倾向与激活DNA-PK的能力相关，尽管两个突变体表明Ku70在DNA-PK激活和IR修复中的作用是分开的。Ku70中DNA-PK自身磷酸化位点和其他结构基序的突变表明，这些位点在体内IR修复中并非必需。这些研究揭示了双链断裂修复所需的Ku70特征。
