Ortega Pedro, Bournique Elodie, Li Junyi, Sanchez Ambrocio, Santiago Gisselle, Harris Brooke R, Green Abby M, Buisson Rémi
Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA, USA.
Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA, USA.
bioRxiv. 2024 Nov 15:2024.11.14.623607. doi: 10.1101/2024.11.14.623607.
ATR is the master safeguard of genomic integrity during DNA replication. Acute inhibition of ATR with ATR inhibitor (ATRi) triggers a surge in origin firing, leading to increased levels of single-stranded DNA (ssDNA) that rapidly deplete all available RPA. This leaves ssDNA unprotected and susceptible to breakage, a phenomenon known as replication catastrophe. However, the mechanism by which unprotected ssDNA breaks remains unclear. Here, we reveal that APOBEC3B is the key enzyme targeting unprotected ssDNA at replication forks, triggering a reaction cascade that induces fork collapse and PARP1 hyperactivation. Mechanistically, we demonstrate that uracils generated by APOBEC3B at replication forks are removed by UNG2, creating abasic sites that are subsequently cleaved by APE1 endonuclease. Moreover, we demonstrate that APE1-mediated DNA cleavage is the critical enzymatic step for PARP1 trapping and hyperactivation in cells, regardless of how abasic sites are generated on DNA. Finally, we show that APOBEC3B-induced toxic PARP1 trapping in response to ATRi drives cell sensitivity to ATR inhibition, creating to a context of synthetic lethality when combined with PARP inhibitors. Together, these findings reveal the mechanisms that cause replication forks to break during replication catastrophe and explain why ATRi-treated cells are particularly sensitive to PARP inhibitors.
ATR是DNA复制过程中基因组完整性的主要保护因子。用ATR抑制剂（ATRi）急性抑制ATR会引发起始点激发激增，导致单链DNA（ssDNA）水平升高，迅速耗尽所有可用的RPA。这使得ssDNA未受保护且易断裂，这种现象称为复制灾难。然而，未受保护的ssDNA断裂的机制仍不清楚。在这里，我们揭示APOBEC3B是靶向复制叉处未受保护的ssDNA的关键酶，引发反应级联，诱导叉形结构崩溃和PARP1过度激活。从机制上讲，我们证明APOBEC3B在复制叉处产生的尿嘧啶被UNG2去除，产生无碱基位点，随后被APE1核酸内切酶切割。此外，我们证明APE1介导的DNA切割是细胞中PARP1捕获和过度激活的关键酶促步骤，无论DNA上的无碱基位点是如何产生的。最后，我们表明APOBEC3B响应ATRi诱导的毒性PARP1捕获导致细胞对ATR抑制敏感，与PARP抑制剂联合使用时产生合成致死的情况。总之，这些发现揭示了复制灾难期间导致复制叉断裂的机制，并解释了为什么用ATRi处理的细胞对PARP抑制剂特别敏感。
