Topoisomerase I (TOP1) resolves DNA topology during replication and transcription. The enzyme forms an intermediate TOP1 cleavage complex (TOP1cc) through transient TOP1–DNA-protein crosslinks. Camptothecin is a frontline anticancer agent that freezes this reaction intermediate, leading to the generation of irreversible TOP1ccs that act as 3′-blocking lesions. It is widely accepted that TOP1cc is repaired via a two-step pathway involving proteasomal degradation of TOP1cc to the crosslinked peptide, followed by removal of the TOP1cc-derived peptide from DNA by tyrosyl-DNA phosphodiesterase 1 (TDP1). In the present study, we developed an assay system to estimate repair kinetics of TOP1cc separately in the first and second steps, using monoclonal antibodies against the TOP1 protein and the TOP1 catalytic site peptide–DNA complex, respectively. Although TDP1-deficient (TDP1^−/−) TK6 cells had normal kinetics of the first step, a delay in the kinetics of the second step was observed relative to that in wild-type cells. Tyrosyl-DNA phosphodiesterase 2 (TDP2) reportedly promotes the repair of TOP1-induced DNA damage in the absence of TDP1. The present assays additionally demonstrated that TDP2 promotes the second, but not the first, step of TOP1cc repair in the absence of TDP1. We also analyzed sensitivities of TK6 cells with deficiencies in TDP1 and/or TDP2 to agents that produce 3′ -blocking lesions. These experiments showed that TDP1^−/−TDP2^−/− cells were more sensitive to the agents Azidothymidine (zidovudine), Cytarabine, Abacavir, Gemcitabine, and Trifluridine than TDP1^−/− or TDP2^−/− cells. Taken together, our findings confirm the roles of TDP2 in the repair of 3′-blocking lesions.

拓扑异构酶I（TOP1）在复制和转录过程中解析DNA拓扑。该酶通过瞬时的TOP1-DNA-蛋白质交联形成中间的TOP1裂解复合物（TOP1cc）。喜树碱是一线抗癌剂，可冻结该反应中间体，从而导致产生不可逆的TOP1cc，这些TOP1cc充当阻断3'的病变。TOP1cc通过一个两步途径，涉及蛋白酶体降解TOP1cc到交联的肽，然后通过酪氨酰DNA磷酸二酯酶1（TDP1）从DNA去除TOP1cc衍生的肽。在本研究中，我们开发了一种测定系统，分别使用针对TOP1蛋白和TOP1催化位点肽-DNA复合体的单克隆抗体分别评估了TOP1cc的修复动力学。尽管缺乏TDP1的（TDP1 ^-/-）TK6细胞第一步的动力学正常，但相对于野生型，观察到第二步的动力学有所延迟细胞。据报道，在不存在TDP1的情况下，酪氨酰DNA磷酸二酯酶2（TDP2）促进了TOP1诱导的DNA损伤的修复。本测定法另外证明，在不存在TDP1的情况下，TDP2促进了TOP1cc修复的第二步而非第一步。我们还分析了TDP1和/或TDP2缺陷的TK6细胞对产生3'阻滞性病变的药物的敏感性。这些实验表明，TDP1 ^{- / -} TDP2 ^{- / -}细胞对药剂叠氮胸苷（齐多夫定），阿糖胞苷，阿巴卡韦，吉西他滨，和三氟胸苷比更敏感TDP1 ^{- / -}或TDP2 ^{- / -}细胞。综上所述，我们的发现证实了TDP2在修复3'阻滞性病变中的作用。