SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition

The proteins Geminin and binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in human cancers and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.蛋白质Geminin（Geminin）及其结合伴侣Cdt1（Cdt1）对于DNA复制的调控至关重要。本研究表明，E3泛素连接酶适配蛋白（E3 ubiquitin ligase adaptor protein）SPOP可在内源水平结合Geminin，并调控DNA复制过程。SPOP可促进Geminin在赖氨酸残基100与127位点发生K27链接型非降解性多泛素化（K27-linked non-degradative poly-ubiquitination）修饰。该多泛素化修饰通过间接阻断Cdt1与MCM蛋白复合物（MCM protein complex）的结合——这一结合是DNA解旋与复制过程所必需的——从而抑制DNA复制过度激活。SPOP在人类癌症中频发突变，并与肿瘤发生（tumorigenesis）密切相关。本研究证实，癌症相关的SPOP突变会损害Geminin的K27链接型多泛素化修饰，进而诱导复制起始点过度激活与重复复制。SPOP突变引发的复制应激（replication stress）会在ATR抑制剂（ATR inhibitor）处理时触发复制灾难（replication catastrophe）与细胞死亡。本研究结果揭示了SPOP通过抑制DNA复制过度激活与基因组不稳定（genome instability）发挥的肿瘤抑制功能，并提示携带SPOP突变的肿瘤可能对ATR抑制剂疗法具有敏感性。