No abstract

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates various aspects of DNA replication. We previously showed that the chromatin around replisomes is rich in SUMO and depleted in Ub, whereas an opposite pattern is observed in mature chromatin. How this SUMO-rich/Ub-low environment is maintained at sites of DNA replication is not known. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Chemical inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced to chromatin away from replisomes. Our findings provide a model to explain the differential accumulation of SUMO and Ub at replication forks, and identify an essential role of USP7 in DNA replication that should be taken into account for the use of USP7 inhibitors as anticancer agents.

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance.

Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands of origins in G1 that are not necessarily activated in the subsequent S-phase. These “dormant” origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality.

The SMC5/6 complex is the least understood of SMC complexes. In yeast, smc5/6 mutants phenocopy mutations in sgs1, the BLM ortholog that is deficient in Bloom's syndrome (BS). We here show that NSMCE2 (Mms21, in Saccharomyces cerevisiae), an essential SUMO ligase of the SMC5/6 complex, suppresses cancer and aging in mice. Surprisingly, a mutation that compromises NSMCE2-dependent SUMOylation does not have a detectable impact on murine lifespan. In contrast, NSMCE2 deletion in adult mice leads to pathologies resembling those found in patients of BS. Moreover, and whereas NSMCE2 deletion does not have a detectable impact on DNA replication, NSMCE2-deficient cells also present the cellular hallmarks of BS such as increased recombination rates and an accumulation of micronuclei. Despite the similarities, NSMCE2 and BLM foci do not colocalize and concomitant deletion of Blm and Nsmce2 in B lymphocytes further increases recombination rates and is synthetic lethal due to severe chromosome mis-segregation. Our work reveals that SUMO-and BLM-independent activities of NSMCE2 limit recombination and facilitate segregation; functions of the SMC5/6 complex that are necessary to prevent cancer and aging in mice.

Polyploidization is a natural process that frequently accompanies differentiation; its deregulation is linked to genomic instability and cancer. Despite its relevance, why cells select different polyploidization mechanisms is unknown. Here we report a systematic genetic analysis of endomitosis, a process in which megakaryocytes become polyploid by entering mitosis but aborting anaphase. Whereas ablation of the APC/C cofactor Cdc20 results in mitotic arrest and severe thrombocytopenia, lack of the kinases Aurora-B, Cdk1, or Cdk2 does not affect megakaryocyte polyploidization or platelet levels. Ablation of Cdk1 forces a switch to endocycles without mitosis, whereas polyploidization in the absence of Cdk1 and Cdk2 occurs in the presence of aberrant re-replication events. Importantly, ablation of these kinases rescues the defects in Cdc20 null megakaryocytes. These findings suggest that endomitosis can be functionally replaced by alternative polyploidization mechanisms in vivo and provide the cellular basis for therapeutic approaches aimed to discriminate mitotic and polyploid cells.

Purpose: There is a lack of prognostic and predictive biomarkers in epithelial ovarian carcinoma, and the targeting of oncogenic signaling pathways has had limited impact on patient survival in this highly heterogeneous disease. The origin licensing machinery, which renders chromosomes competent for DNA replication, acts as a convergence point for upstream signaling pathways. We tested the hypothesis that Cdc7 kinase, a core component of the licensing machinery, is predictive of clinical outcome and may constitute a novel therapeutic target in epithelial ovarian carcinoma. Experimental Design: A total of 143 cases of ovarian cancer and 5 cases of normal ovary were analyzed for Cdc7 protein expression dynamics and clinicopathologic features. To assess the therapeutic potential of Cdc7, expression was down-regulated by RNA interference in SKOV-3 and Caov-3 ovarian cancer cells. Results: Increased Cdc7 protein levels were significantly associated with arrested tumor differentiation (P = 0.004), advanced clinical stage (P = 0.01), genomic instability (P < 0.001), and accelerated cell cycle progression. Multivariate analysis shows that Cdc7 predicts diseasefree survival independent of patient age, tumor grade and stage (hazard ratio, 2.03; confidence interval, 1.53-2.68; P < 0.001), with the hazard ratio for relapse increasing to 10.90 (confidence interval, 4.07-29.17) for the stages 3 to 4/upper Cdc7 tertile group relative to stages 1to 2/lower Cdc7 tertile tumors. In SKOV-3 and Caov-3 cells, Cdc7 siRNA knockdown triggered high levels of apoptosis, whereas untransformed cells arrest in G 1 phase and remain viable. Conclusions: Our findings show that Cdc7 kinase predicts survival and is a potent anticancer target in epithelial ovarian carcinoma, highlighting its potential as a predictor of susceptibility to small molecule kinase inhibitors currently in development.

Edited by Patrick Sung Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPRmediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells. This work was supported by the Spanish Ministry of Economy and Competitiveness and FEDER Grants BFU2013-48481-R and BFU2016-79841-R (to A. L.) and BFU2016-80402-R (to J. M.) and by FPI "Severo Ochoa" fellowships (to C. M. and M. R.-T.). This work was also supported by funding from Boehringer Ingelheim Fonds (to M. R.-T.). The authors declare that they have no conflicts of interest with the contents of this article. Author's Choice-Final version open access under the terms of the Creative Commons CC-BY license. This article contains Figs. S1-S5 and Tables S1 and S2.