DNA–protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2–DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function nonredundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative nonhomologous end joining (NHEJ) pathways. Double-mutant analysis indicates that “clean” DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.

中文翻译：

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拟南芥拓扑异构酶2裂解复合物的修复

必须修复 DNA-蛋白质交联 (DPC) 和 DNA 双链断裂 (DSB)，包括由停滞的拓扑异构酶 2 切割复合物 (TOP2ccs) 产生的那些，以确保基因组稳定性。TOP2cc 修复的基本机制已在其他真核生物中得到表征，但我们缺乏植物的信息。使用 CRISPR/Cas 诱导的突变体，我们表明拟南芥具有两种主要的 TOP2cc 修复途径：一种由水解 TOP2-DNA 键的酪氨酰-DNA-磷酸二酯酶 2 (TDP2) 定义，另一种由 DNA 依赖性蛋白酶 WSS1A 定义（人类 SPARTAN/smt3 [Wss1] 酵母弱抑制因子的同源物），它也在 DPC 修复中起作用。TDP1 和 TDP2 在 TOP1cc 修复中发挥非冗余功能，表明它们特异性地作用于各自停滞的切割复合物。核酸酶甲基磺酸甲酯和紫外线敏感蛋白 81 (MUS81) 在全球 DPC 修复中起主要作用，在 TOP2cc 修复中起次要作用。DSB 作为 TOP2cc 修复的中间体出现，并通过经典和替代的非同源末端连接 (NHEJ) 途径进行修复。双突变分析表明，由 TDP2 水解引起的“干净”DNA 末端主要由经典的 NHEJ 重新连接，这有助于避免突变。相比之下，诱变替代 NHEJ 途径主要处理不可连接的 DNA 末端。因此，TDP2 通过对 TOP2cc 的无错误修复来促进植物基因组完整性的维持。双突变分析表明，由 TDP2 水解引起的“干净”DNA 末端主要由经典的 NHEJ 重新连接，这有助于避免突变。相比之下，诱变替代 NHEJ 途径主要处理不可连接的 DNA 末端。因此，TDP2 通过对 TOP2cc 的无错误修复来促进植物基因组完整性的维持。双突变分析表明，由 TDP2 水解引起的“干净”DNA 末端主要由经典的 NHEJ 重新连接，这有助于避免突变。相比之下，诱变替代 NHEJ 途径主要处理不可连接的 DNA 末端。因此，TDP2 通过对 TOP2cc 的无错误修复来促进植物基因组完整性的维持。