Ubiquitination-dependent DNA damage response (DDR) signals play a critical role in the cellular choice of DNA damage repair pathways. Human DNA helicase RecQL4 participates in DNA replication and repair, and loss of RecQL4 is associated with autosomal recessive genetic disorders characterized by genomic instability features. In an earlier study, RecQL4 was isolated as a stable complex that contained two ubiquitin ligases of the N-end rule (UBR1 and UBR2). However, it is unknown whether or not RecQL4 ubiquitination status is critical for its DNA repair function. Here, we report that RecQL4 directly interacts with RNF8 (a RING finger ubiquitin E3 ligase), and both co-localize at DNA double-strand break (DSB) sites. Our findings indicate that RNF8 ubiquitinates RecQL4 protein mainly at the lysine sites of 876, 1048, and 1101, thereby facilitating the dissociation of RecQL4 from DSB sites. RecQL4 mutant at ubiquitination sites had a significantly prolonged retention at DSBs, which hinders the recruitment of its direct downstream DSB repair proteins (CtIP & Ku80). Interestingly, reduced DSB repair capacity observed in RecQL4 depleted cells was restored only by the reconstitution of wild-type RecQL4, but not the ubiquitination mutant. Additionally, RecQL4 directly interacts with WRAP53β that is known to recruit RNF8 to DSBs and WRAP53β enhances the association of RecQL4 with RNF8. WRAP53β silencing resulted in a nearly diminished recruitment of RNF8 to DSBs and in a greatly attenuated dissociation of RecQL4 from the DSB sites. Collectively, our study demonstrates that the ubiquitination event mediated by RNF8 constitutes an essential component for RecQL4’s function in DSB repair.

泛素依赖性的DNA损伤反应（DDR）信号在DNA损伤修复途径的细胞选择中起着至关重要的作用。人类DNA解旋酶RecQL4参与DNA复制和修复，而RecQL4的丧失与以基因组不稳定特征为特征的常染色体隐性遗传疾病有关。在较早的研究中，RecQL4被分离为稳定的复合物，其中包含两个N端规则的泛素连接酶（UBR1和UBR2）。但是，尚不清楚RecQL4泛素化状态对其DNA修复功能是否至关重要。在这里，我们报告RecQL4直接与RNF8（环指泛素E3连接酶）相互作用，并且都共同定位在DNA双链断裂（DSB）位点。我们的发现表明，RNF8泛素化RecQL4蛋白主要位于876、1048和1101的赖氨酸位点，从而促进RecQL4从DSB站点的解离。RecQL4突变体在泛素化位点在DSB处的保留时间显着延长，这阻碍了其直接下游DSB修复蛋白（CtIP和Ku80）的募集。有趣的是，仅通过野生型RecQL4的重建，而不能通过泛素化突变体的重建，可以恢复在RecQL4耗尽的细胞中观察到的降低的DSB修复能力。此外，RecQL4与已知可将RNF8募集到DSB的WRAP53β直接相互作用，并且WRAP53β增强了RecQL4与RNF8的关联。WRAP53β沉默导致RNF8向DSB的募集几乎减少，并且RecQL4从DSB位点的解离大大减弱。总的来说，