Studying Translesion DNA Synthesis Using Xenopus In Vitro Systems.

Cell-free extracts derived from Xenopus eggs have been widely used to decipher molecular pathways involved in several cellular processes including DNA synthesis, the DNA damage response, and genome integrity maintenance. We set out assays using Xenopus cell-free extracts to study translesion DNA synthesis (TLS), a branch of the DNA damage tolerance pathway that allows replication of damaged DNA. Using this system, we were able to recapitulate TLS activities that occur naturally in vivo during early embryogenesis. This chapter describes protocols to detect chromatin-bound TLS factors by western blotting and immunofluorescence microscopy upon induction of DNA damage by UV irradiation, monitor TLS-dependent mutagenesis, and perform proteomic screening.

Detection of endogenous translesion DNA synthesis in single mammalian cells.

Translesion DNA synthesis (TLS) is an evolutionarily conserved process that cells activate to tolerate DNA damage. TLS facilitates proliferation under DNA damage conditions and is exploited by cancer cells to gain therapy resistance. It has been so far challenging to analyze endogenous TLS factors such as PCNAmUb and TLS DNA polymerases in single mammalian cells due to a lack of suitable detection tools. We have adapted a flow cytometry-based quantitative method allowing detection of endogenous, chromatin-bound TLS factors in single mammalian cells, either untreated or exposed to DNA-damaging agents. This high-throughput procedure is quantitative, accurate, and allows unbiased analysis of TLS factors' recruitment to chromatin, as well as occurrence of DNA lesions with respect to the cell cycle. We also demonstrate detection of endogenous TLS factors by immunofluorescence microscopy and provide insights into TLS dynamics upon DNA replication forks stalled by UV-C-induced DNA damage.

Protocol to analyze endogenous translesion DNA synthesis in single mammalian cells.

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.

Translesion DNA synthesis-driven mutagenesis in very early embryogenesis of fast cleaving embryos.

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.

Studying the DNA damage response in embryonic systems.

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.

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.

Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites.

Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA-protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.

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.

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.

Recent advances in understanding DNA replication: cell type-specific adaptation of the DNA replication program.

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.

Author Correction: RNAs coordinate nuclear envelope assembly and DNA replication through ELYS recruitment to chromatin.

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.

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

Upon 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.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

Half of the human genome is made up 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 in Xenopus laevis egg extract. Using this approach we characterized the 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 including 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 the inability of the 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 for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

Mta2 promotes Tipin-dependent maintenance of replication fork integrity.

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.

DNA replication and homologous recombination factors: acting together to maintain genome stability.

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.

A dynamic history of gene duplications and losses characterizes the evolution of the SPARC family in eumetazoans.

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 to SPARC and SPARCB described 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, SPARC and SPARCB paralogues 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.

The protease degrading sperm histones post-fertilization in sea urchin eggs is a nuclear cathepsin L that is further required for embryo development.

Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp) belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.

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

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.

[Genome sequencing in the sea urchin embryo: what is new concerning the cell cycle?]. / Embryon d'oursin et séquençage du génome de l'espèce S. purpuratus: quels apports à l'étude du cycle cellulaire?

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.

Antimitotic activity of methoxyconidiol, a meroterpene isolated from an ascidian.

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.