拓扑异构酶抑制剂喜树碱(CPT)诱导叉失速,对增殖细胞具有高毒性。然而,细胞对CPT诱导的叉子失速的反应尚未完全表征。这里,我们报道,Cockayne综合征B组(CSB)蛋白抑制了低剂量CPT后PRIMPOL依赖性分叉引发.在高浓度的CPT,CSB需要通过MUS81-RAD52-POLD3依赖性断裂诱导的复制(BIR)来促进DNA复制的重新开始。在没有CSB的情况下,通过POLQ-LIG3-,可以在高浓度的CPT下恢复DNA合成,LIG4-,或PRIMPOL依赖性途径,被抑制的,分别,RAD51、BRCA1和BRCA2蛋白。POLQ和LIG3是替代端连接(Alt-EJ)的核心部件,而LIG4是非同源末端连接(NHEJ)的核心成分。这些结果表明,CSB在高剂量CPT诱导的叉子失速后调节叉子重启途径的选择,促进BIR,但抑制Alt-EJ,NHEJ,和叉子倒灌。我们发现CSB和BRCA2的丢失是在高浓度CPT下基因组稳定性和细胞存活的毒性组合。这可能是由于ssDNA缺口的积累,强调CSB在调节缺乏功能性BRCA2的癌症治疗反应中的重要作用。
Topoisomerase inhibitor camptothecin (CPT) induces fork stalling and is highly toxic to proliferating cells. However, how cells respond to CPT-induced fork stalling has not been fully characterized. Here, we report that Cockayne syndrome group B (CSB) protein inhibits PRIMPOL-dependent fork repriming in response to a low dose of CPT. At a high concentration of CPT, CSB is required to promote the restart of DNA replication through MUS81-RAD52-POLD3-dependent break-induced replication (BIR). In the absence of CSB, resumption of DNA synthesis at a high concentration of CPT can occur through POLQ-LIG3-, LIG4-, or PRIMPOL-dependent pathways, which are inhibited, respectively, by RAD51, BRCA1, and BRCA2 proteins. POLQ and LIG3 are core components of alternative end joining (Alt-EJ), whereas LIG4 is a core component of nonhomologous end joining (NHEJ). These results suggest that CSB regulates fork restart pathway choice following high-dosage CPT-induced fork stalling, promoting BIR but inhibiting Alt-EJ, NHEJ, and fork repriming. We find that loss of CSB and BRCA2 is a toxic combination to genomic stability and cell survival at a high concentration of CPT, which is likely due to accumulation of ssDNA gaps, underscoring an important role of CSB in regulating the therapy response in cancers lacking functional BRCA2.