Study of the G1-phase specific novel repair pathway for X-ray-induced DNA damage

电离辐射（IR）诱导多种DNA损伤。最显著的损伤是DNA双链断裂（DSB），通过同源重组或非同源末端连接（NHEJ）途径修复。由于我们之前证明了IR应答蛋白53BP1特异性地增强了DNA连接酶IV （NHEJ所需的DNA连接酶）的活性，我们研究了53BP1缺陷鸡DT40细胞对IR的反应。53bp1缺陷细胞在G1期对x射线的敏感性增加。虽然内部s和G2/M检查点完好无损，但在53bp1缺陷细胞中辐照后，等染色单体型染色体畸变的频率升高。此外，在53bp1缺失的细胞中，x射线诱导的DNA dsb标记γ-H2AX灶的消失时间延长。因此，G1期细胞的x射线敏感性升高可归因于ir诱导的dna损伤的修复缺陷。显像分析显示53BP1在不同于ku依赖和artemis依赖的NHEJ途径中发挥作用，但需要DNA连接酶IV。引人注目的是，53BP1基因的破坏以及wortmannin对磷脂酰肌醇3-激酶家族的抑制完全消除了G1期照射细胞的集落形成。这些结果表明，53bp1依赖的修复途径对于细胞周期G1期受IR照射的细胞的存活是重要的。

Ionizing radiation (IR) induces a variety of DNA lesions. The most significant lesion is a DNA double-strand break (DSB), which is repaired by homologous recombination or nonhomologous end joining (NHEJ) pathway. Since we previously demonstrated that IR-responsive protein 53BP1 specifically enhances activity of DNA ligase IV, a DNA ligase required for NHEJ, we investigated responses of 53BP1-deficient chicken DT40 cells to IR. 53BP1-deficient cells showed increased sensitivity to X-rays during G1 phase. Although intra-S and G2/M checkpoints were intact, a frequency of isochromatid-type chromosomal aberrations was elevated after irradiation in 53BP1-deficient cells. Furthermore, disappearance of X-ray-induced γ-H2AX foci, a marker of DNA DSBs, was prolonged in 53BP1-deficient cells. Thus, the elevated X-ray sensitivity in G1 phase cells was attributable to repair defect for IR-induced DNA-damage. Epistasis analysis revealed that 53BP1 plays a role in a pathway distinct from the Ku-dependent and Artemis-dependent NHEJ pathways, but requires DNA ligase IV. Strikingly, disruption of the 53BP1 gene together with inhibition of phosphatidylinositol 3-kinase family by wortmannin completely abolished colony formation by cells irradiated during G1 phase. These results demonstrate that the 53BP1-dependent repair pathway is important for survival of cells irradiated with IR during the G1 phase of the cell cycle.