Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4^CSA ubiquitin ligase. How CRL4^CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4^CSA for optimal RNAPII ubiquitylation. Drug–genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.

细胞采用转录偶联修复 (TCR) 来消除转录阻断 DNA 损伤。DNA 损伤诱导的 TCR 特异性修复因子 CSB 与 RNA 聚合酶 II (RNAPII) 的结合通过 CRL4 ^CSA泛素连接酶触发单个赖氨酸 (K1268) 的 RNAPII 泛素化。CRL4 ^CSA如何专门针对 K1268 尚不清楚。在这里，我们将 ELOF1 识别为促进 RNAPII 泛素化的缺失环节，这是组装下游修复因子的关键信号。此功能需要其与靠近 K1268 的 RNAPII 的本构相互作用，揭示 ELOF1 作为结合和定位 CRL4 ^{CSA的特异性因子}以获得最佳的 RNAPII 泛素化。药物-遗传相互作用筛选还揭示了一个 CSB 独立途径，其中 ELOF1 阻止活性基因中的 R 环并保护细胞免受 DNA 复制压力。我们的研究提供了对 TCR 分子机制的重要见解，并提供了转录应激反应和 DNA 复制之间相互作用的遗传框架。