The Trim33 ubiquitin ligase limits E2f4-dependent DNA replication [DSB]. The Trim33 ubiquitin ligase limits E2f4-dependent DNA replication [DSB]

Deregulation of RNA polymerase II (RNAPII) by oncoproteins, such as transcription factor Myc, interferes with DNA replication and is a major source of DNA damage and genomic instability. Ubiquitination is a key pathway controlling RNAPII activity via modification of RNAPII subunits or associated regulatory proteins. We uncover a mechanism for genome maintenance by ubiquitin ligase Trim33 and transcription factor E2f4. We show that Trim33 promotes E2f4 protein turnover, restricting interactions of E2f4 with chromatin and with the Recql DNA helicase. Replicative stress blunts Trim33-dependent regulation, which stimulates Recql recruitment to chromatin and facilitates recovery of DNA synthesis. Deletion of Trim33 triggers chronic recruitment of Recql and accelerates DNA replication under stress, accompanied by compromised DDR signaling and DNA repair. Depletion of Trim33 in Myc-overexpressing cells leads to accumulation of replication-associated DNA damage and delays Myc-driven tumorigenesis. We propose that the Trim33-E2f4-Recql axis provides a mechanism to control DNA replication at transcriptionally active chromatin to maintain genome integrity. Overall design: DSB-capture in Trim33-WT and Trim33-KO cells before and after exposure to hydroxyurea

致癌蛋白（oncoproteins，如转录因子Myc）介导的RNA聚合酶II（RNA polymerase II, RNAPII）调控异常，会干扰DNA复制过程，同时也是DNA损伤与基因组不稳定的重要诱因。泛素化（Ubiquitination）是通过修饰RNAPII亚基或其相关调控蛋白，进而调控RNAPII活性的关键通路。本研究揭示了泛素连接酶（ubiquitin ligase）Trim33与转录因子E2f4参与基因组维持的分子机制。研究证实，Trim33可促进E2f4的蛋白周转，进而限制E2f4与染色质以及Recql DNA解旋酶（Recql DNA helicase）的相互作用。复制应激（Replicative stress）会削弱Trim33介导的调控作用，该调控可促进Recql向染色质的招募，并助力DNA合成的恢复过程。Trim33基因缺失会触发Recql的持续性招募，并在应激状态下加速DNA复制，同时伴随DNA损伤应答（DNA Damage Response, DDR）信号通路受损与DNA修复能力缺陷。在过表达Myc的细胞中敲低Trim33，会导致复制相关DNA损伤的积累，并延缓Myc介导的肿瘤发生过程。本研究提出，Trim33-E2f4-Recql信号轴可通过调控转录活跃染色质区域的DNA复制，从而维持基因组完整性。实验整体设计：在羟基脲（hydroxyurea）处理前后的Trim33野生型（Trim33-WT）与Trim33基因敲除（Trim33-KO）细胞中开展DNA双链断裂捕获（DSB-capture）实验。