Frankenberg-Schwager Marlis, Becker Manuela, Garg Irmgard, Pralle Elke, Wolf Hartmut, Frankenberg Dieter
University of Goettingen, Department of Nuclear Medicine, Goettingen, Germany.
Radiat Res. 2008 Dec;170(6):784-93. doi: 10.1667/RR1375.1.
The purpose of this study was to investigate the cell cycle-dependent role of nonhomologous DNA end joining (NHEJ), conservative homologous recombination (HR), and single-strand annealing (SSA) for the repair of simple DNA double-strand breaks (DSBs) induced by H(2)O(2)-mediated OH radicals in CHO cells. Cells of the cell lines V3 (NHEJ-deficient), irs1SF (HR-deficient) and UV41 (SSA-deficient) and their parental cell line AA8 were exposed to various concentrations of H(2)O(2) in G(1) or S phase of the cell cycle and their colony-forming ability was assayed. In G(1) phase, NHEJ was the most important-if not the only-mechanism to repair H(2)O(2)-mediated DSBs; this was similar to results obtained in a parallel study of more complex DSBs induced by sparsely or densely ionizing radiation. Unlike HR (irs1SF)- and SSA (UV41)-deficient cells, the sensitivity of NHEJ-deficient V3 cells to H(2)O(2) relative to parental AA8 cells in G(1) phase is about 50 times higher compared to 200 kV X rays. This points to a specific role of the catalytic subunit of DNA-PK for efficient NHEJ of H(2)O(2)-mediated DSBs that are located at sites critical for the maintenance of the higher-order structure of cellular DNA, whereas X-ray-induced DSBs are distributed stochastically. Surprisingly, SSA-deficient cells in G(1) phase showed an increased sensitivity to high concentrations of H(2)O(2) relative to the parental wild-type cells and to HR-deficient cells, which may be interpreted in terms of a specific type of H(2)O(2)-induced damage requiring SSA for repair after its transfer into S phase. In S phase, HR is the most important mechanism to repair H(2)O(2)-mediated DSBs, followed by NHEJ. In contrast, the action of error-prone SSA may not be beneficial, since SSA-deficient cells are three times more resistant to H(2)O(2) than wild-type AA8 cells. This is likely due to channeling of DSBs into the error-free HR repair pathway or into the potentially error-prone NHEJ pathway. Cells with or without a defect in DSB repair are considerably more sensitive to H(2)O(2) in S phase compared to G(1) phase. This effect is likely due to the fact that topoisomerase II, which is expressed only in proliferating cells, is a target of H(2)O(2), resulting in enhanced accumulation of DSBs and killing of cells treated in S phase with H(2)O(2). The relative sensitivities to H(2)O(2) differ by orders of magnitude for the four cell lines. This seems to be caused mainly by H(2)O(2)-mediated poisoning of topoisomerase IIalpha rather than by a defect in DSB repair.
本研究的目的是调查非同源DNA末端连接（NHEJ）、保守同源重组（HR）和单链退火（SSA）在细胞周期依赖性修复由H₂O₂介导的OH自由基诱导的简单DNA双链断裂（DSB）中的作用，这些修复发生在CHO细胞中。将V3（NHEJ缺陷型）、irs1SF（HR缺陷型）和UV41（SSA缺陷型）细胞系及其亲本细胞系AA8的细胞在细胞周期的G₁期或S期暴露于不同浓度的H₂O₂，并检测它们的集落形成能力。在G₁期，NHEJ是修复H₂O₂介导的DSB的最重要机制——即便不是唯一机制；这与在一项关于由稀疏或密集电离辐射诱导的更复杂DSB的平行研究中获得的结果相似。与HR（irs1SF）缺陷型和SSA（UV41）缺陷型细胞不同，NHEJ缺陷型V3细胞在G₁期相对于亲本AA8细胞对H₂O₂的敏感性比200 kV X射线高约50倍。这表明DNA-PK的催化亚基对于有效修复位于对维持细胞DNA高阶结构至关重要的位点的H₂O₂介导的DSB具有特定作用，而X射线诱导的DSB是随机分布的。令人惊讶的是，G₁期的SSA缺陷型细胞相对于亲本野生型细胞和HR缺陷型细胞对高浓度H₂O₂表现出更高的敏感性，这可以解释为一种特定类型的H₂O₂诱导损伤在转移到S期后需要SSA进行修复。在S期，HR是修复H₂O₂介导的DSB的最重要机制，其次是NHEJ。相比之下，易错的SSA的作用可能并无益处，因为SSA缺陷型细胞对H₂O₂的抗性是野生型AA8细胞的三倍。这可能是由于DSB被引导至无错的HR修复途径或可能易错的NHEJ途径。与G₁期相比，有或没有DSB修复缺陷的细胞在S期对H₂O₂的敏感性要高得多。这种效应可能是由于仅在增殖细胞中表达的拓扑异构酶II是H₂O₂的靶点，导致DSB积累增加以及用H₂O₂处理S期细胞时细胞死亡。这四种细胞系对H₂O₂的相对敏感性相差几个数量级。这似乎主要是由H₂O₂介导的拓扑异构酶IIα中毒引起的，而不是由DSB修复缺陷导致的。
