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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 ubiquitinmediated 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., Ramón y Cajal Fellowship from MINECO (RYC-2016-20705), co-funded by European Regional Development Funds (FEDER) to Emilio Lecona; by grants from the Spanish Ministry of Science, Innovation and Universities (RTI2018-102204-B-I00, co-financed with European FEDER funds) and the European Research Council (ERC-617840) to Oscar Fernandez-Capetillo; fellowships from Fundacion Ramon Areces-UAM and LaCaixa Foundation to Pablo Valledor (LCF/BQ/ES18/11670008)

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., Spanish Ministry of Science, Innovation and Universities (RTI2018-102204-B-I00, co-financed with European FEDER funds) and the European Research Council (ERC-617840) to OF; a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-095582-B-I00, co-financed with European FEDER funds) to MM; a grant from MINECO (BFU2014-55168-JIN)

The aim of this work was to analyze outer membrane porin-encoding genes (ompK35 and ompK36) in a collection of OXA-48 producing Klebsiella pneumoniae, to assess the effect of porin alterations on the susceptibility to ceftazidime/avibactam, and to describe a screening methodology for phenotypic detection of OXA-48-producing K. pneumoniae with disrupted porins. Antimicrobial susceptibility was tested by Microscan and Etest. The genomes of 81 OXA-48-producing K. pneumoniae were sequenced. MLST, detection of antimicrobial resistance genes, and analysis of ompK35 and ompK36 were performed in silico. Tridimensional structures of the OmpK36 variants were assessed. Receiver operating characteristics curves were built to visualize the performance ability of a disk diffusion assay using carbapenems and cefoxitin to detect OmpK36 functional alterations. A wide variety of OmpK36 alterations were detected in 17 OXA-48-producing K. pneumoniae isolates. All displayed a high-level meropenem resistance (MIC ≥ 8 mg/L), and some belonged to high-risk clones, such as ST15 and ST147. Alterations in ompK35 were also observed, but they did not correlate with high-level meropenem resistance. All isolates were susceptible to ceftazidime/avibactam and porin alterations did not affect the MICs of the latter combination. Cefoxitin together with ertapenem/meropenem low inhibition zone diameters (equal or lower than 16 mm) could strongly suggest alterations affecting OmpK36 in OXA-48-producing K. pneumoniae. OXA-48-producing K. pneumoniae with porin disruptions are a cause of concern, ceftazidime/avibactam showed good in vitro activity against them, so this combination could be positioned as the choice therapy to combat the infections caused by this difficult-to-treat isolates.

Summary The AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin., Graphical abstract, Highlights • VCP and USP7 control the SUMO/ubiquitin landscape during DNA replication • VCP is recruited by FAF1 to chromatin-associated SUMO/ubiquitylated proteins • FAF1 recognizes both SUMO and ubiquitin on target proteins • The control of DNA replication by VCPFAF1 and USP7 is evolutionarily conserved, Chromatin at DNA replication forks is SUMO-rich and ubiquitin-poor. Franz et al. report that FAF1 is a dual reader of SUMO and ubiquitin targeting VCP to extract DNA replication factors from chromatin in C. elegans and human cells. Together with USP7, VCPFAF1 controls the SUMO/ubiquitin landscape during replication.

SUMMARYTo 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.