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.

Protection from replicative stress conferred by Chk1 promotes transformation.

In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2 TG ) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.Supplemental material is available for this article.Received December 5, 2014; revised version accepted March 2, 2015.Replication stress (RS) has emerged as a source of genome instability in human diseases, including cancer and premature aging (Lecona and Fernandez-Capetillo 2014;Zeman and Cimprich 2014). In brief, RS is defined by the accumulation of abnormal amounts of ssDNA at stalled replication forks that, due to its recombinogenic nature, can initiate genomic rearrangements. In mammals, RS is sensed and suppressed by a signaling cascade initiated by the ATR kinase, which, together with its target kinase, CHK1, suppresses RS through the phosphorylation of multiple targets (Cimprich and Cortez 2008;Lopez-Contreras and Fernandez-Capetillo 2010). ATR and CHK1 are essential for embryonic development in mice (Brown and Baltimore 2000;de Klein et al. 2000;Liu et al. 2000), which is due to the role of the RS response (RSR) in preventing replication-born chromosome breakage. Whether the RSR protects all forks or a subset of them during replication is unclear. On the one hand, proteomic studies of the human replisome in unchallenged conditions have failed to detect ATR or CHK1 in the vicinity of replication forks Sirbu et al. 2013), suggesting that their activity might be particularly necessary for only a subset of forks, such as damaged ones. Accordingly, chromosomal breaks that arise upon ATR inactivation locate preferentially at specific loci named common fragile sites (CFSs) (Casper et al. 2002) and early replicating fragile sites (ERFSs) (Barlow et al. 2013). Regardless of whether ATR works at all forks or only some of them, how it suppresses RS and why it is essential are still not fully understood.Ribonucleotide reductase (RNR) is a tetrameric enzyme composed of two catalytic (RRM1, Rnr1 in yeast) and two regulatory (RRM2, Rnr2 in yeast) subunits (Jordan and Reichard 1998). It reduces NDPs into dNDPs, which is a rate-limiting step for the production of dNTPs. In yeast, the lethality of mec1Δ strains can be bypassed by mutations that increase RNR activity. The first evidence of a connection between ATR and RNR came from the discovery of Crt1 (a transcriptional repressor of RNR subunits) as a suppressor of Mec1 lethality in Saccharomyces cerevisiae (Huang et al. 1998). Furthermore, overproduction of Rnr1 was shown to be sufficient ...

Replicative stress (RS) is a type of endogenous DNA damage that cells suffer every time they duplicate their genomes, and which is further boosted by oncogenes. In mammals, the RS response (RSR) is coordinated by ATR and Chk1 kinases. We sought to develop a mammalian organism that is selectively protected from RS. To this end, mice carrying an extra copy of the Chk1 gene were generated. In vitro, Chk1 transgenic cells are protected from RS-inducing agents. Moreover, an extra Chk1 allele prolongs the survival of ATR-Seckel mice, which suffer from high levels of RS, but not that of ATM-deficient mice, which accumulate DNA breaks. Surprisingly, increased Chk1 levels favor transformation, which we show is associated with a reduction in the levels of RS induced by oncogenes. Our study provides the first example where supra-physiological levels of a tumor suppressor can promote malignant transformation, which is a result of the protection from the RS found in cancer cells.

SummaryDamaged DNA has a profound impact on mammalian health and overall survival. In addition to being the source of mutations that initiate cancer, the accumulation of toxic amounts of DNA damage can cause severe developmental diseases and accelerate ageing. Therefore, understanding how cells respond to DNA damage has become one of the most intense areas of biomedical research in the recent years. However, whereas most mechanistic studies derive from in vitro or in cellulo work, the impact of a given mutation on a living organism is largely unpredictable. For instance, why BRCA1 mutations preferentially lead to breast cancer whereas mutations compromising mismatch repair drive colon cancer is still not understood. In this context, evaluating the specific physiological impact of mutations that compromise genome integrity has become crucial for a better dimensioning of our knowledge. We here describe the various technologies that can be used for modeling mutations in mice, and provide a review of the genes and pathways that have been modeled so far in the context of DNA damage responses.