Highlights d VCP and USP7 control the SUMO/ubiquitin landscape during DNA replication d VCP is recruited by FAF1 to chromatin-associated SUMO/ ubiquitylated proteins d FAF1 recognizes both SUMO and ubiquitin on target proteins d The control of DNA replication by VCP FAF1 and USP7 is evolutionarily conserved

Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53-independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.

Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin–CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.

KEYWORDS: USP7; CDK1; DNA REPLICATION; MITOSIS; S/M TRANSITION. USP7 coordinates the S/M transition 2 Galarreta et al. SUMMARY To ensure a faithful segregation of chromosomes, DNA must be fully replicated before mitotic entry. However, how cells sense the completion of DNA replication and to what extent this is linked to the activation of the mitotic machinery remains poorly understood. We previously showed that USP7 is a replisome-associated deubiquitinase with an essential role in DNA replication. Here, we reveal that USP7 inhibition leads to the ubiquitination of MCM7, a hallmark of DNA replication termination. In addition, USP7 inhibition leads to the ubiquitination of additional replisome components such as POLD1, which are displaced from replisomes. Surprisingly, this premature termination of DNA replication occurs concomitant to a generalized activation of CDK1 throughout the entire cell cycle, which impairs chromosome segregation and is toxic for mammalian cells. Accordingly, the toxicity of USP7 inhibitors is alleviated by CDK1 inhibition. Our work sheds light into the mechanism of action of USP7 inhibitors and provides evidence to the concept that DNA replication termination is coupled to the activation of the mitotic program. USP7 coordinates the S/M transition 3 Galarreta et al.