The involvement of acidic nucleoplasmic DNA-binding protein (and-1) in the regulation of prereplicative complex (pre-RC) assembly in human cells

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2–7 onto chromatin during late mitosis of the cell cycle. MCM2–7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G(1) phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2–7 to facilitate the assembly of MCM2–7 onto chromatin at replication origins in late mitosis and G(1) phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G(1) phase cells. Thus, human And-1 facilitates loading of the MCM2–7 helicase onto chromatin during the assembly of pre-RC

Loss of Cyclin C or CDK8 provides ATR inhibitor resistance by suppressing transcription-associated replication stress.

The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR-Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death

Decreased MCM2-6 in Drosophila S2 cells does not generate significant DNA damage or cause a marked increase in sensitivity to replication interference.

A reduction in the level of some MCM proteins in human cancer cells (MCM5 in U20S cells or MCM3 in Hela cells) causes a rapid increase in the level of DNA damage under normal conditions of cell proliferation and a loss of viability when the cells are subjected to replication interference. Here we show that Drosophila S2 cells do not appear to show the same degree of sensitivity to MCM2-6 reduction. Under normal cell growth conditions a reduction of >95% in the levels of MCM3, 5, and 6 causes no significant short term alteration in the parameters of DNA replication or increase in DNA damage. MCM depleted cells challenged with HU do show a decrease in the density of replication forks compared to cells with normal levels of MCM proteins, but this produces no consistent change in the levels of DNA damage observed. In contrast a comparable reduction of MCM7 levels has marked effects on viability, replication parameters and DNA damage in the absence of HU treatment

Analyse de la dynamique de réplication préméiotique chez Saccharomyces cerevisiae par peignage moléculaire de l'ADN

Chez les eukaryotes, la duplication des chromosomes est initiée à partir des origines de réplication selon une chorégraphie spatio-temporelle bien définie qui contribue au bon déroulement des autres évènements du cycle cellulaire, assurant ainsi la transmission correcte du patrimoine génétique. Ce programme spatio-temporel n'est pas rigide et peut varier selon le type cellulaire et s'adapter aux conditions physiologiques. Chez la levure Saccharomyces cerevisiae, par exemple, la méiose est un exemple de différentiation cellulaire au cours de laquelle une cellule diploïde va générer quatre cellules haploïdes génétiquement différentes, et où la phase de réplication des chromosomes est deux à trois fois plus longue que lors du cycle végétatif. Mon travail de thèse a consisté principalement à définir les causes de cette extension, en utilisant la technique très résolutive du peignage moléculaire de l'ADN qui permet, à partir de molécules uniques, de déterminer la cinétique d'activation des origines et la progression des fourches de réplication. L'analyse d'un chromosome isolé (Chr.VI) indique que les mêmes origines sont utilisées en mitose et méiose, mais selon un programme d'activation différent. Nos résultats suggèrent aussi qu'en méiose les fourches de réplication ralentiraient au niveau de sites de pause. J'ai alors tenté d'élucider les causes moléculaires de ces pauses et leur lien éventuel avec la mise en place de la recombinaison méiotique, une étape fondamentale pour la ségrégation correcte des chromosomes et le brassage génétique lors de la formation des gamètes. Ce processus est initié par la formation des Cassures Double Brin (CDB) qui requiert l'intervention d'un complexe protéique spécifique de la méiose, constitué notamment de Mer2, Rec114 et Spo11. La dynamique de réplication a été étudiée dans des souches mutées pour ces protéines. Parallèlement à ces travaux sur la méiose, j'ai aussi amélioré la technique du peignage moléculaire et démontré son utilité pour déterminer avec précision la fin de la réplication des chromosomes, une donnée qui n'était pas accessible par les techniques classiques. J'ai ainsi pu montrer que des cellules dépourvues en Cdc14, une protéine phosphatase essentielle pour la ségrégation de l'ADN ribosomique (rDNA) et la sortie de mitose, terminaient la réplication plus tardivement que les cellules contrôles. Le fort retard de réplication du rDNA dans le mutant cdc14-1 pourrait être responsable de son défaut de ségrégation en anaphaseThe duplication of chromosomes in eukaryotes initiates from numerous origins that are activated during S phase according to specific spatio-temporal replication programs. These replication programs are connected to downstream cell cycle events and contribute to accurate transmission of the genetic material to progeny, yet they are flexible and can adapt to varying physiological conditions. In the yeast Saccharomyces cerevisiae, for example, meiosis can be considered as a differentiation program whereby a diploid cell gives rise to four genetically different haploid cells. Interestingly, premeiotic DNA replication is usually two to three times longer than during vegetative cell division (mitosis), in multiple organisms, yet no one really knows why. The aim of my thesis work was to uncover the reasons for this S phase extension in meiosis, using a state-of-the-art imaging technique called DNA combing. With this technique that I contributed to improve, the firing of origins as well as replication fork progression rates can be monitored on the level of single DNA molecules. My data indicate that the same number of origins is used in mitosis and meiosis. However, by focusing on a single chromosome (Chr.VI) we discovered that, although the same set of origins is used, it is activated following a different program. A first subset of origins fires with high efficiency, then replication forks seem to pause for a long while before a second subset of origins fires. I tried using various mutants to determine the nature of these replication pausing sites and their potential link with the induction of meiotic recombination, which is essential for correct chromosome segregation in meiosis. This process begins with the formation of double-strand breaks (DSBs) that require the concerted action of a number of meiotic-specific proteins, among which Mer2, Rec114 and Spo11. In order to see if these DSB proteins are responsible the lengthening of S in meiosis, I analyzed replication dynamics in strains lacking these proteins. Besides this work, I also demonstrated the utility of DNA combing for defining when DNA replication is completed in mitotic cells, a measure that was not available from current techniques. This way I was able to show that yeast cdc14-1 cells, defective for a conserved protein phosphatase needed for ribosomal DNA (rDNA) segregation and mitotic exit, finish rDNA replication much later than control cells. It is likely that the failure of cdc14-1 cells to finish rDNA replication in time is responsible for its non-segregation in anaphaseMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

The Function of the Coding Sequences for the Putative Pheromone Precursors in Podospora anserina Is Restricted to Fertilization

We cloned the pheromone precursor genes of Podospora anserina in order to elucidate their role in the biology of this fungus. The mfp gene encodes a 24-amino-acid polypeptide finished by the CAAX motif, characteristic of fungal lipopeptide pheromone precursors similar to the a-factor precursor of Saccharomyces cerevisiae. The mfm gene encodes a 221-amino-acid polypeptide, which is related to the S. cerevisiae α-factor precursor and contains two 13-residue repeats assumed to correspond to the mature pheromone. We deleted the mfp and mfm coding sequence by gene replacement. The mutations specifically affect male fertility, without impairing female fertility and vegetative growth. The male defect is mating type specific: the mat+ Δmfp and mat− Δmfm mutants produce male cells inactive in fertilization whereas the mat− Δmfp and mat+ Δmfm mutants show normal male fertility. Genetic data indicate that both mfp and mfm are transcribed at a low level in mat+ and mat− vegetative hyphae. Northern-blot analysis shows that their transcription is induced by the mating types in microconidia (mfp by mat+ and mfm by mat−). We managed to cross Δmfp Δmfm strains of opposite mating type, by complementation and transient expression of the pheromone precursor gene to trigger fertilization. These crosses were fertile, demonstrating that once fertilization occurs, the pheromone precursor genes are unnecessary for the completion of the sexual cycle. Finally, we show that the constitutively transcribed gpd::mfm and gpd::mfp constructs are repressed at a posttranscriptional level by the noncognate mating type

Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis.

Proliferating trophoblast stem cells (TSCs) can differentiate into nonproliferating but viable trophoblast giant cells (TGCs) that are resistant to DNA damage induced apoptosis. Differentiation is associated with selective up-regulation of the Cip/Kip cyclin-dependent kinase inhibitors p57 and p21; expression of p27 remains constant. Previous studies showed that p57 localizes to the nucleus in TGCs where it is essential for endoreplication. Here we show that p27 also remains localized to the nucleus during TSC differentiation where it complements the role of p57. Unexpectedly, p21 localized to the cytoplasm where it was maintained throughout both the G- and S-phases of endocycles, and where it prevented DNA damage induced apoptosis. This unusual status for a Cip/Kip protein was dependent on site-specific phosphorylation of p21 by the Akt1 kinase that is also up-regulated in TGCs. Although cytoplasmic p21 is widespread among cancer cells, among normal cells it has been observed only in monocytes. The fact that it also occurs in TGCs reveals that p57 and p21 serve nonredundant functions, and suggests that the role of p21 in suppressing apoptosis is restricted to terminally differentiated cells

The p27 and p57 proteins are localized to the nucleus in G-phase TGCs.

<p>After three days of FGF4 deprivation, wild-type TGCs were cultured for 20 min in the presence of EdU (10 µM) to label S-phase cells. After fixation, EdU was detected using Click-iT chemistry (green), and the cells were stained with either anti-p57 (cyan) (A, C) or anti-p27 (red) (B, C) antibodies, and with Hoechst 33342 to visualize nuclear DNA (gray). Images were acquired with a confocal microscope (63x objective). The bottom panels are higher magnifications of merged images in order to visualize differential staining of EdU, p27 and p57. Scale bars represent 50 µm.</p

Akt1 is required for p21 stabilization in TGCs, and Akt1 phosphorylates p21 at T140.

<p>(A) C-terminal sequence of mouse p21 protein with the putative Akt1 phosphorylation site (RKRRQTS) and the essential RKR nuclear localization signal <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097434#pone.0097434-RodriguezVilarrupla1" target="_blank">[44]</a> in bold face. (B) Extracts of TSCs and TGCs were assayed by Western immuno-blotting using antibodies specific for Akt1, p21, p27 and p57 proteins. The phosphorylated form of p21 was recognized as a p21 protein that migrated slower than unphosphorylated p21, and by its reaction with an anti-p21 antibody specific for Thr-145 phosphorylation in human p21. (C) After three days of FGF4 deprivation, TGCs were stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize nuclear DNA (gray) and with anti-Akt1 antibody (green). (D) Wild-type p21 and two p21 mutant forms with a T140V or a S141A substitution were tested as substrates for phosphorylation by Akt1 <i>in vitro</i>. (E) NIH3T3 fibroblasts were co-transfected with a plasmid expressing the tetracycline repressor and a plasmid encoding the indicated p21 protein whose expression was regulated by a tetracycline inducible promoter. Each protein carried a [His]₆-cMyc-epitope tag fused to its C-terminus. After 24 hours of transfection, the cells were cultured for 18 hours in the presence of tetracycline (1 µg/ml) in order to induce expression of the indicated recombinant p21 protein. Cells were then harvested at 0, 6, 12 and 24 hours after release, and extracts were analyzed for the indicated protein by Western immuno-blotting using a Myc-Tag specific antibody. Wild-type (wt) p21 and the T140V (T/V) and S141A (S/A) p21 mutants, as well as a double mutant (T/V+S/A) were tested. Actin served as a loading control in each case.</p

Target Sequences for shRNAs.

<p>All three shRNAs were packaged into a single lentivirus preparation.</p

Primers used for site-directed mutagenesis of the murine p21 cDNA.

<p>underlined bases are mutated.</p