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In early embryogenesis of fast cleaving embryos, DNA synthesis is short and surveillance mechanisms preserving genome integrity are inefficient, implying the possible generation of mutations. We have analyzed mutagenesis in Xenopus laevis and Drosophila melanogaster early embryos. We report the occurrence of a high mutation rate in Xenopus and show that it is dependent upon the translesion DNA synthesis (TLS) master regulator Rad18. Unexpectedly, we observed a homology-directed repair contribution of Rad18 in reducing the mutation load. Genetic invalidation of TLS in the pre-blastoderm Drosophila embryo resulted in reduction of both the hatching rate and single-nucleotide variations on pericentromeric heterochromatin in adult flies. Altogether, these findings indicate that during very early Xenopus and Drosophila embryos TLS strongly contributes to the high mutation rate. This may constitute a previously unforeseen source of genetic diversity contributing to the polymorphisms of each individual with implications for genome evolution and species adaptation., Graphical Abstract Graphical AbstractSpeculative model of early Xenopus embryo replisome. High Rad18 abundance induces both constitutive PCNA monoubiquitination (PCNAmUb) and active translesion DNA synthesis (TLS) generating mutations (red triangles) at high rate (+ sign). Both Rad18 homology-directed repair activity (HDR) and the mismatch repair system (MMR) function synergistically with TLS to reduce the mutation load (− sign). The Rad18 residues important for TLS activity (C28) and HDR activity (C207) are also indicated.

Maintenance and surveillance of genome integrity is crucial during the very early steps of embryonic development, since de novo mutations generated during this stage can be propagated in differentiated adult cells and may lead to predisposition to diseases including cancer. Surprisingly, early embryos are characterized by a relaxed control of genome integrity, reminiscent of that observed in cancer cells. How embryos manage to produce healthy adult individuals in such conditions remains still unclear. Here, we describe protocols and methods to study and analyze the DNA damage response and genome integrity in two embryonic experimental systems, early Xenopus laevis embryos and mouse embryonic stem cells. We describe methods to study gene functions in the DNA damage response by mRNA microinjection in Xenopus embryos generated by in vitro fertilization, mutagenesis and developmental regulation of the DNA damage response. We also describe methods to analyze the DNA damage response in mESCs, including synchronization experiments that allow studying the DNA damage response at different cell cycle stages. Analysis of genome integrity in these systems may also help to shed light on the molecular mechanisms that preserve genome integrity and become dysregulated in cancer cells.

Maintenance and surveillance of genome integrity is crucial during the very early steps of embryonic development, since de novo mutations generated during this stage can be propagated in differentiated adult cells and may lead to predisposition to diseases including cancer. Surprisingly, early embryos are characterized by a relaxed control of genome integrity, reminiscent of that observed in cancer cells. How embryos manage to produce healthy adult individuals in such conditions remains still unclear. Here, we describe protocols and methods to study and analyze the DNA damage response and genome integrity in two embryonic experimental systems, early Xenopus laevis embryos and mouse embryonic stem cells. We describe methods to study gene functions in the DNA damage response by mRNA microinjection in Xenopus embryos generated by in vitro fertilization, mutagenesis and developmental regulation of the DNA damage response. We also describe methods to analyze the DNA damage response in mESCs, including synchronization experiments that allow studying the DNA damage response at different cell cycle stages. Analysis of genome integrity in these systems may also help to shed light on the molecular mechanisms that preserve genome integrity and become dysregulated in cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

International audience; During the very early stages of embryonic development chromosome replication occurs under rather challenging conditions, including a very short cell cycle, absence of transcription, a relaxed DNA damage response and, in certain animal species, a highly contracted S-phase. This raises the puzzling question of how the genome can be faithfully replicated in such a peculiar metabolic context. Recent studies have provided new insights into this issue, and unveiled that embryos are prone to accumulate genetic and genomic alterations, most likely due to restricted cellular functions, in particular reduced DNA synthesis quality control. These findings may explain the low rate of successful development in mammals and the occurrence of diseases, such as abnormal developmental features and cancer. In this review, we will discuss recent findings in this field and put forward perspectives to further study this fascinating question.

Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation., Origins of replications are associated with potential G quadruplexes forming structures (G4s). Here the authors reveal the functional role of G4 elements in DNA replication initiation.

Orderly progression of S phase requires the action of replisome-associated Tipin and Tim1 proteins, whose molecular function is poorly understood. Here, we show that Tipin deficiency leads to the accumulation of aberrant replication intermediates known as reversed forks. We identified Mta2, a subunit of the NuRD chromatin remodeler complex, as a novel Tipin binding partner and mediator of its function. Mta2 is required for Tipin-dependent Polymerase α binding to replicating chromatin, and this function is essential to prevent the accumulation of reversed forks. Given the role of the Mta2-NuRD complex in the maintenance of heterochromatin, which is usually associated with hard-to-replicate DNA sequences, we tested the role of Tipin in the replication of such regions. Using a novel assay we developed to monitor replication of specific genomic loci in Xenopus laevis egg extract we demonstrated that Tipin is directly required for efficient replication of vertebrate centromeric DNA. Overall these results suggest that Mta2 and Tipin cooperate to maintain replication fork integrity, especially on regions that are intrinsically difficult to duplicate.

International audience; Half of human genome is made of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using Bacterial Artificial Chromosomes (BACs) in Xenopus laevis egg extract. Using this approach we characterized chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication dependent enrichment of a network of DNA repair factors among which the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to inability of single stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of Topoisomerase I dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications on our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

DNA replication in metazoan cells initiates at multiple discrete chromosomal sites called replication origins. Recent genome-wide studies have mapped thousands of origins in animal and plant cells, but without yielding a distinct and universal consensus sequence. However, origin-associated regions with particular base composition features have been identified, such as the G-rich OGRE motif, predicted to form G-quadruplexes. Epigenetic marks such as histone modifications that promote open chromatin also favor origin formation.

In the original version of this Article, the affiliation details for Antoine Aze, Michalis Fragkos, Stéphane Bocquet, Julien Cau and Marcel Méchali incorrectly omitted ‘CNRS and the University of Montpellier’. This has now been corrected in both the PDF and HTML versions of the Article.

Sea urchin is a classical research model system in developmental biology; moreover, the external fertilization and growth of embryos, their rapid division cycle, their transparency and the accessibility of these embryos to molecular visualization methods, made them good specimens to analyze the regulatory mechanisms of cell division. These features as well as the phylogenetic position of sea urchin, close to vertebrates but in an outgroup within the deuterostomes, led scientists working on this model to sequence the genome of the species S. purpuratus. The genome contains a full repertoire of cell cycle control genes. A comparison of this toolkit with those from vertebrates, nematodes, drosophila, as well as tunicates, provides new insight into the evolution of cell cycle control. While some gene subtypes have undergone lineage-specific expansions in vertebrates (i.e. cyclins, mitotic kinases,...), others seem to be lost in vertebrates, for instance the novel cyclin B identified in S. purpuratus. On the other hand, some genes which were previously thought to be vertebrate innovations, are also found in sea urchins (i.e. MCM9). To note is also the absence of cell cycle inhibitors of the INK type, which are apparently confined to vertebrates. The uncovered genomic repertoire of cell-cycle regulators will thus provide molecular tools that should further enhance future research on cell cycle control and developmental regulation in this model.

International audience; A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model.

Genome duplication requires the coordinated action of multiple proteins to ensure a fast replication with high fidelity. These factors form a complex called the Replisome, which is assembled onto the DNA duplex to promote its unwinding and to catalyze the polymerization of two new strands. Key constituents of the Replisome are the Cdc45-Mcm2-7-GINS helicase and the And1-Claspin-Tipin-Tim1 complex, which coordinate DNA unwinding with polymerase alpha-, delta-, and epsilon- dependent DNA polymerization. These factors encounter numerous obstacles, such as endogenous DNA lesions leading to template breakage and complex structures arising from intrinsic features of specific DNA sequences. To overcome these roadblocks, homologous recombination DNA repair factors, such as Rad51 and the Mre11-Rad50-Nbs1 complex, are required to ensure complete and faithful replication. Consistent with this notion, many of the genes involved in this process result in lethal phenotypes when inactivated in organisms with complex and large genomes. Here, we summarize the architectural and functional properties of the Replisome and propose a unified view of DNA replication and repair processes.

Fertilization relieves the oocyte from a cell cycle arrest, inducing progression towards mitotic cycles. While the signalling pathways involved in oocyte to embryo transition have been widely investigated, how they specifically trigger DNA replication is still unclear. We used sea urchin eggs whose oocytes are arrested in G1 to investigate in vivo the molecular mechanisms regulating initiation of replication after fertilization. Unexpectedly, we found that CDC6, Cdt1 and MCM3, components of the pre-replication complexes (pre-RC) which license origins for replication, were already loaded on female chromatin before fertilization. This is the first demonstration of a cell cycle arrest in metazoan in which chromatin is already licensed for replication. In contrast pre-RC assemble on chromatin post-fertilization as in other organisms. These differences in the timing of pre-RC assembly are accompanied by differences in Cdk2 requirement for DNA replication initiation between female and male chromatin post-fertilization. Finally, we demonstrated that a concomitant inhibition of MAP kinase and ATM/ATR pathways releases the block to DNA synthesis. Our findings provide new insight into the mechanisms contributing to the release of G1 arrest and the control of S-phase entry at fertilization.

From oogenesis through fertilization and gastrulation, embryos use various mechanisms to regulate cell expansion, keeping a strict balance between cell proliferation, cell differentiation and cell death. While rapid divisions are necessary at the initial stage to ensure early embryo survival, further developmental transitions are marked by changes in cell cycle and transcriptional regulation. Pluripotency and capability of self-renewal are maintained in a low percentage of cells, the embryonic stem cells (ESCs), which will be later used as a cellular source for tissue replacement. Clearly, some essential characteristics of cell cycle and transcriptional regulation of the early embryo will be conserved in ESC lines. We addressed here the peculiarities of these developmental programs in early embryos and pluripotent embryonic cell lines, considering examples from marine invertebrates to mammals. Finally, we discussed the importance of transcription regulation and chromatin remodelling and their peculiar features in embryonic cells from these species.

Sea urchin is a classical research model system in developmental biology; moreover, the external fertilization and growth of embryos, their rapid division cycle, their transparency and the accessibility of these embryos to molecular visualization methods, made them good specimens to analyze the regulatory mechanisms of cell division. These features as well as the phylogenetic position of sea urchin, close to vertebrates but in an outgroup within the deuterostomes, led scientists working on this model to sequence the genome of the species S. purpuratus. The genome contains a full repertoire of cell cycle control genes. A comparison of this toolkit with those from vertebrates, nematodes, drosophila, as well as tunicates, provides new insight into the evolution of cell cycle control. While some gene subtypes have undergone lineage-specific expansions in vertebrates (i.e. cyclins, mitotic kinases,...), others seem to be lost in vertebrates, for instance the novel cyclin B identified in S. purpuratus. On the other hand, some genes which were previously thought to be vertebrate innovations, are also found in sea urchins (i.e. MCM9). To note is also the absence of cell cycle inhibitors of the INK type, which are apparently confined to vertebrates. The uncovered genomic repertoire of cell-cycle regulators will thus provide molecular tools that should further enhance future research on cell cycle control and developmental regulation in this model.

Methoxyconidiol is a meroterpene previously extracted from the ascidian Aplidium aff. densum [A. Simon-Levert, A. Arrault, N. Bontemps-Subielos, C. Canal, B. Banaigs. Meroterpenes from the ascidian Aplidium aff. densum, J. Nat. Prod. 68 (2005) 1412–1415]. In the present work we investigated its antimitotic effect on eukaryotic cells by using a bioassay based on the sea urchin early embryo. This bioassay has been successfully used to evaluate the efficacy of antiproliferative agents and to rapidly determine the affected cell cycle phase. We demonstrated that methoxyconidiol inhibits the cleavages of sea urchin Sphaerechinus granularis and Paracentrotus lividus fertilized eggs. This meroterpene disrupts M-phase progression and completely blocks cytokinesis without having any effect on DNA replication. The treatment severely disturbs the establishment of a mitotic spindle, most likely by affecting microtubule dynamics. Moreover, while the cell cycle regulatory kinase cyclin B/CDK1 is activated, cyclin B proteolysis is inhibited, impeding the output of M-phase. This characteristic cell cycle arrest induced by methoxyconidiol in sea urchin eggs emphasizes the interest for this drug as a putative antiproliferative agent for tumor cells.

The vertebrates share the ability to produce a skeleton made of mineralized extracellular matrix. However, our understanding of the molecular changes that accompanied their emergence remains scarce. Here, we describe the evolutionary history of the SPARC (secreted protein acidic and rich in cysteine) family, because its vertebrate orthologues are expressed in cartilage, bones and teeth where they have been proposed to bind calcium and act as extracellular collagen chaperones, and because further duplications of specific SPARC members produced the small calcium-binding phosphoproteins (SCPP) family that is crucial for skeletal mineralization to occur. Both phylogeny and synteny conservation analyses reveal that, in the eumetazoan ancestor, a unique ancestral gene duplicated to give rise toSPARCandSPARCBdescribed here for the first time. Independent losses have eliminated one of the two paralogues in cnidarians, protostomes and tetrapods. Hence, only non-tetrapod deuterostomes have conserved both genes. Remarkably,SPARCandSPARCBparalogues are still linked in the amphioxus genome. To shed light on the evolution of the SPARC family members in chordates, we performed a comprehensive analysis of their embryonic expression patterns in amphioxus, tunicates, teleosts, amphibians and mammals. Our results show that in the chordate lineage SPARC and SPARCB family members were recurrently recruited in a variety of unrelated tissues expressing collagen genes. We propose that one of the earliest steps of skeletal evolution involved the co-expression of SPARC paralogues with collagenous proteins.