We recently reported that when low doses of ionizing radiation induce low numbers of DNA double-strand breaks (DSBs) in G₂-phase cells, about 50 % of them are repaired by homologous recombination (HR) and the remaining by classical non-homologous end-joining (c-NHEJ). However, with increasing DSB-load, the contribution of HR drops to undetectable (at ∼10 Gy) as c-NHEJ dominates. It remains unknown whether the approximately equal shunting of DSBs between HR and c-NHEJ at low radiation doses and the predominant shunting to c-NHEJ at high doses, applies to every DSB, or whether the individual characteristics of each DSB generate processing preferences. When G₂-phase cells are irradiated, only about 10 % of the induced DSBs break the chromatids. This breakage allows analysis of the processing of this specific subset of DSBs using cytogenetic methods. Notably, at low radiation doses, these DSBs are almost exclusively processed by HR, suggesting that chromatin characteristics awaiting characterization underpin chromatid breakage and determine the preferential engagement of HR. Strikingly, we also discovered that with increasing radiation dose, a pathway switch to c-NHEJ occurs in the processing of this subset of DSBs. Here, we confirm and substantially extend our initial observations using additional methodologies. Wild-type cells, as well as HR and c-NHEJ mutants, are exposed to a broad spectrum of radiation doses and their response analyzed specifically in G₂ phase. Our results further consolidate the observation that at doses <2 Gy, HR is the main option in the processing of the subset of DSBs generating chromatid breaks and that a pathway switch at doses between 4–6 Gy allows the progressive engagement of c-NHEJ. PARP1 inhibition, irrespective of radiation dose, leaves chromatid break repair unaffected suggesting that the contribution of alternative end-joining is undetectable under these experimental conditions.

我们最近报道，当低剂量的电离辐射在 G _{2 期}细胞中诱导少量 DNA 双链断裂 (DSBs) 时，其中约 50% 通过同源重组 (HR) 修复，其余通过经典非同源重组端接 (c-NHEJ)。然而，随着 DSB 负荷的增加，由于 c-NHEJ 占主导地位，HR 的贡献下降到不可检测（约 10 Gy）。尚不清楚在低辐射剂量下 HR 和 c-NHEJ 之间 DSB 的分流大致相等，而在高剂量下主要分流到 c-NHEJ，是否适用于每个 DSB，或者每个 DSB 的个体特征是否会产生处理偏好。当 G ₂期细胞被照射，只有大约 10% 的诱导 DSB 破坏染色单体。这种断裂允许使用细胞遗传学方法分析这个特定的 DSB 子集的处理。值得注意的是，在低辐射剂量下，这些 DSB 几乎完全由 HR 处理，这表明等待表征的染色质特征支持染色单体断裂并决定 HR 的优先参与。引人注目的是，我们还发现随着辐射剂量的增加，在处理该 DSB 子集时会发生向 c-NHEJ 的途径转换。在这里，我们使用其他方法确认并大幅扩展了我们的初步观察。野生型细胞，以及 HR 和 c-NHEJ 突变体，暴露在广谱的辐射剂量下，并在 G _{2 中}专门分析了它们的反应阶段。我们的结果进一步巩固了以下观察结果，即在剂量 <2 Gy 时，HR 是处理产生染色​​单体断裂的 DSB 子集的主要选择，并且在 4-6 Gy 剂量之间的通路切换允许 c-NHEJ 的逐步参与。无论辐射剂量如何，PARP1 抑制都不会影响染色单体断裂修复，这表明在这些实验条件下无法检测到替代末端连接的贡献。