Zhang Ying, Zhou Junqing, Cao Xiaofan, Zhang Qinming, Lim Chang U K, Ullrich Robert L, Bailey Susan M, Liber Howard L
Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80521, USA.
Cancer Lett. 2007 May 18;250(1):63-73. doi: 10.1016/j.canlet.2006.09.021. Epub 2006 Nov 13.
The correct repair of DNA double-strand breaks (DSBs) is essential to maintaining the integrity of the genome. Misrepair of DSBs is detrimental to cells and organisms, leading to gene mutation, chromosomal aberration, and cancer development. Nonhomologous end-joining (NHEJ) is one of the principal rejoining processes in most higher eukaryotic cells. NHEJ is facilitated by DNA-dependent protein kinase (DNA-PK), which is composed of a catalytic subunit, DNA-PKcs, and the heterodimeric DNA binding regulatory complex Ku70/86. Null mutation of DNA-PKcs leads to immunodeficiency, chromosomal aberration, gene mutation, telomeric end-capping failure, and cancer predisposition in animals and cells. However, it is unknown whether partial deficiency of DNA-PKcs as might occur in a fraction of the population (e.g., heterozygotes), influences cellular function. Using small interfering RNA (siRNA) transfection, we established partial deficiency of DNA-PKcs in human cells, ranging from 4 to 85% of control levels. Our results reveal for the first time, that partial deficiency of DNA-PKcs leads to increased ionizing radiation (IR)-induced mutagenesis, cell killing, and telomere dysfunction. Radiation mutagenesis was increased inversely with DNA-PKcs protein level, with the most pronounced effect being observed in cells with protein levels below 50% of controls. A small but statistically significant increase in IR-induced cell killing was observed as DNA-PKcs levels decreased, over the entire range of protein levels. Frequencies of IR-induced telomere-DSB fusion was increased at levels of DNA-PKcs as low as approximately 50%, similar to what would be expected in heterozygous individuals. Taken together, our results suggest that even partial deficiency of DNA repair proteins may represent a considerable risk to genomic stability.
DNA双链断裂（DSB）的正确修复对于维持基因组的完整性至关重要。DSB修复错误对细胞和生物体有害，会导致基因突变、染色体畸变和癌症发生。非同源末端连接（NHEJ）是大多数高等真核细胞中的主要重新连接过程之一。DNA依赖性蛋白激酶（DNA-PK）促进NHEJ，它由一个催化亚基DNA-PKcs和异源二聚体DNA结合调节复合物Ku70/86组成。DNA-PKcs的无效突变会导致动物和细胞出现免疫缺陷、染色体畸变、基因突变、端粒末端加帽失败和癌症易感性。然而，DNA-PKcs的部分缺陷（如在一部分人群中可能出现的杂合子情况）是否会影响细胞功能尚不清楚。通过小干扰RNA（siRNA）转染，我们在人类细胞中建立了DNA-PKcs的部分缺陷，其水平在对照水平的4%至85%之间。我们的结果首次揭示，DNA-PKcs的部分缺陷会导致电离辐射（IR）诱导的诱变增加、细胞杀伤增加和端粒功能障碍。辐射诱变与DNA-PKcs蛋白水平呈负相关，在蛋白水平低于对照50%的细胞中观察到最明显的影响。随着DNA-PKcs水平下降，在整个蛋白水平范围内，IR诱导的细胞杀伤均有小幅但具有统计学意义的增加。IR诱导的端粒-DSB融合频率在DNA-PKcs水平低至约50%时增加，类似于杂合个体中预期的情况。综上所述，我们的结果表明，即使是DNA修复蛋白的部分缺陷也可能对基因组稳定性构成相当大的风险。
