Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK.
The University of Sheffield Neuroscience Institute and the Healthy Lifespan Institute, Department of Molecular Biology and Biotechnology, Firth Court, University of Sheffield, S10 2TN, Sheffield, UK.
Nat Commun. 2020 Mar 9;11(1):1274. doi: 10.1038/s41467-020-15000-w.
Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate-a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc)-is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase (TDP1), which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3' phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. Here we find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution.
真核拓扑异构酶 1（TOP1）调节 DNA 拓扑结构，以确保有效的 DNA 复制和转录。TOP1 也是内源性基因组不稳定性的主要驱动因素，尤其是当其催化中间体——一种称为 TOP1 断裂复合物（TOP1cc）的共价 TOP1-DNA 加合物——稳定时。TOP1ccs 具有高度细胞毒性，并且如果不能将其解决，就会导致神经紊乱的病理学，但也被用于癌症治疗，其中 TOP1ccs 是广泛使用的一线抗癌药物的靶标。TOP1cc 解决的关键酶是酪氨酸-DNA 磷酸二酯酶（TDP1），它水解将 TOP1 活性位点中的酪氨酸与单链（ss）DNA 断裂处的 3'磷酸基团连接起来的键。然而，TDP1 只能处理来自 ssDNA 末端的小肽片段，这就提出了一个问题，即如何在上游于 TDP1 处处理约 90 kDa 的 TOP1 蛋白。在这里，我们发现 TEX264 通过与 p97 ATPase 和 SPRTN 金属蛋白酶形成复合物来履行这一作用。我们表明，TEX264 识别未经修饰和 SUMO1 修饰的 TOP1，并通过招募 p97 和 SPRTN 启动 TOP1cc 修复。TEX264 定位于核周，与 DNA 复制叉相关联，并在 DNA 复制过程中对抗 TOP1ccs。总之，我们的研究阐明了存在一个专门的修复复合物，该复合物需要用于 TOP1ccs 的上游蛋白水解及其随后的解决。
