Department of Radiation Oncology and Radiation Therapy, University Medical Centre of the Johannes Gutenberg, University Mainz, Germany.
Department of Radiation Oncology and Radiation Therapy, University Medical Centre of the Johannes Gutenberg, University Mainz, Germany.
DNA Repair (Amst). 2020 Dec;96:102992. doi: 10.1016/j.dnarep.2020.102992. Epub 2020 Oct 6.
Fanconi anemia (FA) is a rare chromosomal instability syndrome with various clinical features and high cancer incidence. Despite being a DNA repair disorder syndrome and a frequently observed clinical hypersensitivity of FA patients towards ionizing radiation, the experimental evidence regarding the efficiency of radiation-induced DNA double-strand break (DSB) repair in FA is very controversial. Here, we performed a thorough analysis of the repair of radiation-induced DSBs in G1 and G2 in FA fibroblasts of complementation groups A, C, D1 (BRCA2), D2, E, F, G and P (SLX4) in comparison to normal human lung and skin fibroblasts. γH2AX, 53BP1, or RPA foci quantification after X-irradiation was combined with cell cycle markers. Cytogenetic analyses were performed on first metaphases after irradiation in G1 and by premature chromosome condensation after exposure in G2. Furthermore, the role of canonical-NHEJ and alternative-NHEJ for the fidelity of the repair of radiation-induced DSBs was examined. In FA fibroblasts, DSB repair was normal in G1 but compromised and more error-prone in the slow repair component of G2 as suggested by higher yields of radiation-induced γH2AX and 53BP1 foci as well as chromatid exchanges. However, RPA foci quantification in G2 indicated proficiency for homology-directed repair of DSBs in FA except for FA D1 (BRCA2). In lung fibroblasts, DSB repair in G1 was conducted with normal kinetics but elevated chromosome exchanges compared to skin fibroblasts. The overall repair of radiation-induced DSBs and the formation of chromosome exchanges in normal and FA fibroblasts in G1 and G2 were governed by canonical-NHEJ with no contribution of alternative-NHEJ. Together, we show impaired repair of radiation-induced DSBs in various FA complementation groups in the slow repair component of G2 that might promote the formation of potentially oncogenic aberrations and clinical radiation hypersensitivity.
范可尼贫血（FA）是一种罕见的染色体不稳定综合征，具有多种临床特征和高癌症发病率。尽管 FA 是一种 DNA 修复障碍综合征，并且 FA 患者经常表现出对电离辐射的临床超敏反应，但关于 FA 中辐射诱导的 DNA 双链断裂（DSB）修复效率的实验证据存在很大争议。在这里，我们对补体组 A、C、D1（BRCA2）、D2、E、F、G 和 P（SLX4）的 FA 成纤维细胞中的 G1 和 G2 中辐射诱导的 DSB 修复进行了全面分析，与正常人类肺和皮肤成纤维细胞进行了比较。X 射线照射后 γH2AX、53BP1 或 RPA 焦点的定量与细胞周期标志物相结合。G1 中进行了第一次有丝分裂前的细胞遗传学分析，G2 中进行了过早染色体凝聚后的细胞遗传学分析。此外，还研究了经典非同源末端连接（NHEJ）和替代性 NHEJ 对辐射诱导的 DSB 修复保真度的作用。在 FA 成纤维细胞中，G1 中的 DSB 修复正常，但在 G2 的缓慢修复成分中受损且更容易出错，这表明更高的辐射诱导 γH2AX 和 53BP1 焦点以及染色单体交换的产量。然而，G2 中的 RPA 焦点定量表明 FA 中除 FA D1（BRCA2）外，同源定向修复 DSB 的能力。在肺成纤维细胞中，与皮肤成纤维细胞相比，G1 中的 DSB 修复具有正常的动力学，但染色体交换增加。正常和 FA 成纤维细胞在 G1 和 G2 中整体修复辐射诱导的 DSB 以及形成染色体交换受经典 NHEJ 控制，替代性 NHEJ 没有贡献。总之，我们发现各种 FA 补体组在 G2 的缓慢修复成分中辐射诱导的 DSB 修复受损，这可能促进潜在致癌异常和临床辐射超敏反应的形成。
