DNA end resection is needed for the repair of complex lesions in G1-phase human cells

ABSTRACTRepair of DNA double strand breaks (DSBs) is influenced by the chemical complexity of the lesion. Clustered lesions (complex DSBs) are generally considered more difficult to repair and responsible for early and late cellular effects after exposure to genotoxic agents. Resection is commonly used by the cells as part of the homologous recombination (HR) pathway in S- and G2-phase. In contrast, DNA resection in G1-phase may lead to an error-prone microhomology-mediated end joining. We induced DNA lesions with a wide range of complexity by irradiation of mammalian cells with X-rays or accelerated ions of different velocity and mass. We found replication protein A (RPA) foci indicating DSB resection both in S/G2- and G1-cells, and the fraction of resection-positive cells correlates with the severity of lesion complexity throughout the cell cycle. Besides RPA, Ataxia telangiectasia and Rad3-related (ATR) was recruited to complex DSBs both in S/G2- and G1-cells. Resection of complex DSBs is driven by meiotic recombination 11 homolog A (MRE11), CTBP-interacting protein (CtIP), and exonuclease 1 (EXO1) but seems not controlled by the Ku heterodimer or by phosphorylation of H2AX. Reduced resection capacity by CtIP depletion increased cell killing and the fraction of unrepaired DSBs after exposure to densely ionizing heavy ions, but not to X-rays. We conclude that in mammalian cells resection is essential for repair of complex DSBs in all phases of the cell-cycle and targeting this process sensitizes mammalian cells to cytotoxic agents inducing clustered breaks, such as in heavy-ion cancer therapy.

摘要：DNA双链断裂（DSBs）的修复受损伤化学复杂性的影响。成簇损伤（复杂DSBs）通常被认为更难以修复，并负责在接触致突变剂后早期和晚期的细胞效应。细胞在S期和G2期通常通过同源重组（HR）途径使用切除作为修复的一部分。相反，G1期的DNA切除可能导致易错的微同源性介导的末端连接。我们通过使用不同速度和质量的X射线或加速离子对哺乳细胞进行照射，诱导了具有广泛复杂性的DNA损伤。我们发现，复制蛋白A（RPA）在S/G2-和G1细胞中均指示DSB切除，且切除阳性细胞的比率与细胞周期中损伤复杂性的严重程度相关。除了RPA外，Ataxia telangiectasia and Rad3-related（ATR）也在S/G2-和G1细胞中被招募到复杂DSBs。复杂DSBs的切除由减数分裂重组11同源A（MRE11）、CTBP相互作用蛋白（CtIP）和核酸外切酶1（EXO1）驱动，但似乎不受Ku异二聚体或H2AX磷酸化的控制。CtIP耗竭降低切除能力，增加了细胞在接触密集电离重离子后的细胞杀伤和未修复DSBs的比率，但不是X射线。我们得出结论，在哺乳细胞中，切除对于细胞周期所有阶段的复杂DSBs修复是至关重要的，并且针对这一过程可以提高哺乳细胞对诱导成簇断裂的细胞毒性剂（如重离子癌症疗法）的敏感性。