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（DNA double strand breaks, DSBs）的修复受损伤的化学复杂性调控。成簇损伤（complex DSBs）通常被认为修复难度更高，且是细胞暴露于遗传毒性剂后引发早期与晚期细胞效应的关键诱因。在S期与G2期的细胞中，核酸切除（DNA resection）常作为同源重组（homologous recombination, HR）通路的核心组分发挥作用。与之相反，G1期的DNA核酸切除可能会引发易出错的微同源介导末端连接（microhomology-mediated end joining）。本研究通过用X射线或不同速度、不同质量的加速离子照射哺乳动物细胞，诱导出复杂度跨度广泛的DNA损伤。研究发现，复制蛋白A（replication protein A, RPA）灶点可作为DSB切除的特异性标记，该现象同时存在于S/G2期与G1期细胞中，且切除阳性细胞的比例在整个细胞周期中与损伤复杂度的严重程度呈正相关。除RPA外，毛细血管扩张共济失调与Rad3相关激酶（Ataxia telangiectasia and Rad3-related, ATR）也会被招募至S/G2期与G1期细胞内的复杂DSBs损伤位点。复杂DSBs的核酸切除由减数分裂重组11同源物A（meiotic recombination 11 homolog A, MRE11）、CtBP相互作用蛋白（CTBP-interacting protein, CtIP）与外切酶1（exonuclease 1, EXO1）介导驱动，但似乎不受Ku异二聚体（Ku heterodimer）或H2AX磷酸化的调控。通过CtIP基因敲低降低核酸切除能力，会提升细胞在暴露于高密度电离重离子后的致死率与未修复DSBs的比例，但对X射线照射后的细胞无此类影响。本研究得出结论：在哺乳动物细胞中，核酸切除过程对于细胞周期各阶段的复杂DSBs修复均不可或缺，靶向该通路可使哺乳动物细胞对诱导成簇断裂的细胞毒性剂更为敏感，这一策略可应用于重离子癌症治疗等场景。