Recent advances in understanding DNA replication: cell type–specific adaptation of the DNA replication program [version 1; referees: 2 approved]

DNA replication is an essential process occurring prior to cell division. Cell division coupled to proliferation ensures the growth and renewal of a large variety of specialized cell types generated during embryonic development. Changes in the DNA replication program occur during development. Embryonic undifferentiated cells show a high replication rate and fast proliferation, whereas more differentiated cells are characterized by reduced DNA synthesis and a low proliferation rate. Hence, the DNA replication program must adapt to the specific features of cells committed to different fates. Recent findings on DNA synthesis regulation in different cell types open new perspectives for developing efficient and more adapted therapies to treat various diseases such as genetic diseases and cancer. This review will put the emphasis on recent progress made in this field

Replication origins are already licensed in G1 arrested unfertilized sea urchin eggs

AbstractFertilization 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

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus

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

Involvement of G-quadruplex regions in mammalian replication origin activity.

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

Author Correction: Involvement of G-quadruplex regions in mammalian replication origin activity

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

Preserving Genome Integrity during the Early Embryonic DNA Replication Cycles

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

Protocol to analyze endogenous translesion DNA synthesis in single mammalian cells

Summary: Translesion DNA synthesis (TLS) is an evolutionarily conserved branch of the cellular DNA damage tolerance pathway that is often exploited by cancer cells to overcome therapy resistance. Here, we present a protocol to analyze endogenous TLS in single mammalian cells in the absence or presence of DNA damage. We describe steps for detecting chromatin-bound TLS factors, such as monoubiquitinated PCNA(mUb) and TLS DNA polymerases (pols) by flow cytometry. We then detail a procedure to detect their nuclear localization using immunofluorescence.For complete details on the use and execution of this protocol, please refer to Egger et al. (Cell Reports Methods, in press).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Etude des événements précoces qui suivent la fécondation et conditionnent le cycle de division mitotique

La reproduction sexuée repose sur deux processus essentiels : la méiose où les gamètes sont produits, et la fécondation, caractérisée par la fusion des gamètes mâle et femelle pour former le zygote. Chez la plupart des organismes, la fécondation libère les œufs d un arrêt méiotique et induit la progression vers les événements mitotiques précoces. Alors que les voies de signalisations impliquées dans cette transition ont été largement étudiées, la façon dont elles déclenchent l initiation de la synthèse d ADN reste mal comprise. La synthèse de l ADN est initiée au niveau d origines de réplication (ori), qui acquièrent leur compétence à répliquer suite au recrutement de protéines constituant le complexe de pré-réplication (pré-RC). Le recrutement de Cdt1 est- régulé par son interaction avec géminine et l activation des oris est contrôlée par le jeu de phosphorylations impliquant différentes kinases. Les oeufs d oursin sont arrêtés à l état haploïde en phase G1 du cycle cellulaire. Ils constituent un avantage particulier pour étudier in vivo la régulation de la réplication de l ADN puisque cette étape suit directement la fécondation. Nous avons montré que les pré-RC sont formés sur la chromatine mâle post-fécondation alors qu ils sont déjà assemblés sur la chromatine femelle dans les œufs arrêtés en G1. Il s agit de la première démonstration d un arrêt du cycle cellulaire chez les métazaoires où la chromatine est déjà compétente à répliquer l ADN. Nous avons ensuite étudié le rôle des voies de signalisation dans le maintien et la libération du blocage du cycle. Enfin, nous avons abordé les mécanismes de régulation des composants du pre-RC durant le cycle cellulaire.PARIS-BIUSJ-Physique recherche (751052113) / SudocBANYULS/MER-Observ.Océanol. (660162201) / SudocSudocFranceF

Embryon d'oursin et séquençage du génome de l'espèce

Depuis longtemps, l'oursin est un modèle privilégié pour les recherches sur le développement. De plus, la fécondation et la croissance externe des embryons, leur cycle de division rapide ainsi que leur transparence, appropriée aux techniques de visualisation moléculaire les plus actuelles, font également de l'embryogenèse précoce de l'oursin un modèle de choix pour l'analyse des mécanismes qui régulent la division cellulaire. Ces caractéristiques, ainsi que la position phylogénétique de l'oursin, proche des vertébrés et cependant dans un groupe externe, ont conduit la communauté scientifique travaillant sur ce modèle à séquencer très récemment le génome complet de l'espèce Strongylo-centrotus purpuratus. Ce génome contient un répertoire pratiquement complet des gènes régulateurs de la division cellulaire. La comparaison avec les répertoires équivalents de vertébrés, d'insectes, de nématodes ou de tuniciers donne un nouvel éclairage à l'évolution du contrôle du cycle cellulaire. Chaque famille de gènes comporte chez l'oursin un nombre limité de composants. Chez les vertébrés, de nombreuses familles ont subi une forte expansion (cyclines, kinases mitotiques...), néanmoins d'autres gènes semblent être absents, comme par exemple une nouvelle cycline de type B mise en évidence chez l'oursin. Par ailleurs, certains gènes, que l'on croyait jusque là spécifiques des vertébrés, existent également chez S. purpuratus (MCM9,...). Enfin, il est important de noter l'absence chez l'oursin des inhibiteurs du cycle cellulaire de la famille des INK, qui seraient donc vraisemblablement spécifiques des vertébrés. Cet apport considérable de nouveaux outils moléculaires pour l'oursin donnera sans aucun doute un nouvel essor aux travaux concernant les mécanismes de division dans cette espèce

RNAs coordinate nuclear envelope assembly and DNA replication through ELYS recruitment to chromatin

International audienceUpon fertilisation, the sperm pronucleus acquires the competence to replicate the genome through a cascade of events that link chromatin remodelling to nuclear envelope formation. The factors involved have been partially identified and are poorly characterised. Here, using Xenopus laevis egg extracts we show that RNAs are required for proper nuclear envelope assembly following sperm DNA decondensation. Although chromatin remodelling and pre-replication complex formation occur normally, RNA-depleted extracts show a defect in pre-RC activation. The nuclear processes affected by RNA-depletion included ELYS recruitment, which accounts for the deficiency in nuclear pore complex assembly. This results in failure in chromatin relaxation as well as in the import and proper nuclear concentration of the S-phase kinases necessary for DNA replication activation. Our results highlight a translation-independent RNA function necessary for the parental genome progression towards the early embryonic cell cycle programme