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

电离辐射（IR）可引起多种DNA损伤。最显著的损伤是DNA双链断裂（DSB），其通过同源重组或非同源末端连接（NHEJ）途径修复。由于我们以前证明，IR响应蛋白53 BP 1特异性增强的DNA连接酶IV，NHEJ所需的DNA连接酶的活性，我们研究了53 BP 1-缺陷的鸡DT 40细胞的反应IR。53 BP 1-缺陷的细胞表现出增加的敏感性，X射线在G1期。虽然内S和G2/M检查点是完整的，在53 BP 1缺陷细胞照射后，同染色单体型染色体畸变的频率升高。此外，在53 BP 1缺陷细胞中，X射线诱导的γ-H2 AX灶（DNA DSB的标志物）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。上位性分析显示，53 BP 1在不同于Ku依赖性和Artemis依赖性NHEJ途径的途径中起作用，但需要DNA连接酶IV。引人注目的是，53 BP 1基因的破坏与磷脂酰肌醇3-激酶家族的抑制渥曼青霉素完全废除了G1期照射的细胞的集落形成。这些结果表明，53 BP 1依赖性修复途径是重要的细胞周期的G1期期间与IR照射的细胞的存活。

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.