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（double-strand breaks, DSBs）的修复过程受损伤化学复杂性的调控。簇状损伤（complex DSBs，即复杂双链断裂）通常被认为更难修复，且与遗传毒性剂暴露后引发的早期及晚期细胞效应密切相关。细胞通常在S期和G2期将DNA末端切除作为同源重组（homologous recombination, HR）通路的一环。与之相反，G1期的DNA末端切除则可能引发易出错的微同源介导的末端连接。我们通过用X射线或不同速率、不同质量的加速离子辐照哺乳动物细胞，诱导出了一系列复杂度各异的DNA损伤。研究发现，复制蛋白A（replication protein A, RPA）聚焦形成的病灶可指示S/G2期及G1期细胞中的DSB末端切除事件，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，毛细血管扩张性共济失调突变和Rad3相关激酶（Ataxia telangiectasia and Rad3-related, ATR）也会在S/G2期及G1期的细胞中被招募至复杂DSB位点。复杂DSB的末端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）及核酸外切酶1（exonuclease 1, EXO1）介导，但似乎不受Ku异二聚体（Ku heterodimer）或H2AX磷酸化的调控。通过敲低CtIP降低末端切除能力，会增加细胞在暴露于高密度电离重离子后的死亡率及未修复DSB的比例，但对X射线辐照后的细胞无此影响。我们据此得出结论：在哺乳动物细胞中，末端切除对于细胞周期各阶段复杂DSB的修复均至关重要，且靶向该过程可使哺乳动物细胞对诱导簇状断裂的细胞毒性制剂敏感，例如重离子癌症治疗中的应用。