Cdk2 Activity Is Dispensable for the Onset of DNA Replication during the First Mitotic Cycles of the Sea Urchin Early Embryo

Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and inXenopusextracts. In the present report we analyzein vivothe involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. UnfertilizedSphaerechinus granulariseggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studyingin vivothe onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained inXenopusegg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with thisin vitrosystem in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21^Cip1does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensablein vivofor the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast toXenopusegg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.     

Requirement of mouse BCCIP for neural development and progenitor proliferation

Multiple DNA repair pathways are involved in the orderly development of neural systems at distinct stages. The homologous recombination (HR) pathway is required to resolve stalled replication forks and critical for the proliferation of progenitor cells during neural development. BCCIP is a BRCA2 and CDKN1A interacting protein implicated in HR and inhibition of DNA replication stress. In this study, we determined the role of BCCIP in neural development using a conditional BCCIP knock-down mouse model. BCCIP deficiency impaired embryonic and postnatal neural development, causing severe ataxia, cerebral and cerebellar defects, and microcephaly. These development defects are associated with spontaneous DNA damage and subsequent cell death in the proliferative cell populations of the neural system during embryogenesis. With in vitro neural spheroid cultures, BCCIP deficiency impaired neural progenitor's self-renewal capability, and spontaneously activated p53. These data suggest that BCCIP and its anti-replication stress functions are essential for normal neural development by maintaining an orderly proliferation of neural progenitors.     

Subcellular localization of DNA polymerase gamma and changes in its activity in sea urchin embryos

1. Subcellular localization and changes in the activity of DNA polymerase gamma were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The gamma-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase gamma) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.     

Regulation of replication of λ phage and λ plasmid DNAs at low temperature

It was previously demonstrated that while lysogenic development of bacteriophage λ in Escherichia coli proceeds normally at low temperature (20–25° C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the p _E promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20° C. Contrary to previous reports, it is possible to demonstrate that p _E promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20° C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage λ is neither inhibited at 20° C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between λ phage and λ plasmid DNA at low temperature. Received: 30 December 1997 / Accepted: 25 February 1998     

UV irradiation inhibits initiation of DNA replication from oriC in Escherichia coli

Summary Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication. This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect). However, by introducing an unirradiated E. coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon. We therefore conclude that UV-irradiation of E. coli leads to a trans-acting inhibition of initiation of replication. The inhibition is transient and does not appear to be an SOS function.     

Identification of Genomic Regions Required for DNA Replication during Drosophila Embryogenesis

A collection of Drosophila deficiency stocks was examined by bromodeoxyuridine (BrdU) labeling of embryos to analyze the DNA replication patterns in late embryogenesis. This permitted us to screen 34% of the genome for genes that when absent in homozygous deficiencies affect the cell cycle or DNA replication. We found three genomic intervals that when deleted result in cessation of DNA replication in the embryo, 39D2-3;E2-F1, 51E and 75C5-7;F1. Embryos deleted for the 75C5-7;F1 region stop DNA replication at the time in embryogenesis when a G(1) phase is added to the mitotic cell cycle and the larval tissues begin to become polytene. Thus, this interval may contain a gene controlling these cell cycle transitions. DNA replication arrests earlier in embryos homozygous for deletions for the other two regions. Analysis of the effects of deletions in the 39D2-3;E2-F1 region on DNA replication showed that the block to DNA replication correlates with deletion of the histone genes. We were able to identify a single, lethal complementation group in 51E, l(2)51Ec, that is responsible for the cessation of replication observed in this interval. Deficiencies that removed one of the Drosophila cdc2 genes and the cyclin A gene had no effect on replication during embryogenesis. Additionally, our analysis identified a gene, pimples, that is required for the proper completion of mitosis in the post-blastoderm divisions of the embryo.     

Selective Inhibition by Pantoyl Lactone and Butyl Alcohol of the Initiation of DNA Replication in E. coli

We studied action mechanisms of pantoyl lactone and butyl alcohol on the macromolecular synthesis of E. coli. Protein synthesis was not significantly suppressed by these agents. DNA synthesis was more remarkably affected than RNA synthesis by them. Synchronous cultures of E. coli were subsequently used to investigate the inhibition of DNA replication with these agents. It was consequently shown that these agents inhibit the initiation of a new cycle of DNA replication in this organism but permit the completion of DNA replication initiated before addition of these agents to the medium.     

Cloning of Drosophila MCM homologs and analysis of their requirement during embryogenesis

MCM (minichromosome maintenance) gene family of Saccharomyces cerevisiae encodes essential DNA replication factors that participate in the initiation of DNA replication. In addition, their localization to the nucleus in a mitosis-dependent manner fueled the hypothesis that MCMs also act to couple DNA replication to mitosis. We report the identification of a Drosophila gene family with extensive sequence identity to the MCM genes. Results from antibody injection experiments suggest that MCMs play an essential role in DNA replication during embryogenesis. Evolutionary conservation of MCM sequences and function in Drosophila could potentially facilitate studies of how initiation of DNA replication is regulated and coupled to mitosis during metazoan development.     

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection

Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.     

Biogenesis of mitochondria

Summary The action of ethidium bromide and berenil on the mitochondrial genome of Saccharomyces cerevisiae has been compared in three types of study: (i) early kinetics (up to 4 h) of petite induction by the drugs in the presence or absence of sodium dodecyl sulphate; (ii) genetic consequences of long-term (8 cell generations) exposure to the drugs; (iii) inhibition of mitochondrial DNA replication, both in whole cells and in isolated mitochondria.The results have been interpreted as follows. Firstly, the early events in petite induction differ markedly for the two drugs, as indicated by differences in the short-term kinetics. After some stage a common pathway is apparently followed because the composition of the population of petite cells induced after long-term exposure are very similar for both ethidium bromide and berenil. Secondly, both drugs probably act at the same site to inhibit mitochondrial DNA replication, in view of the fact that a petite strain known to be resistant to ethidium bromide inhibition of mitochondrial DNA replication was found to have simultaneously acquired resistance to berenil. From consideration of the drug concentrations needed to inhibit mitochondrial DNA replication in vivo and in vitro it is suggested that in vivo permeability barriers impede the access of ethidium bromide to the site of inhibition of mitochondrial DNA replication, whilst access of berenil to this site is facilitated. The site at which the drugs act to inhibit mitochondrial DNA replication may be different from the site(s) involved in early petite induction. Binding of the drugs at the latter site(s) is considered to initiate a series of events leading to the fragmentation of yeast mitochondrial DNA and petite induction.     

Inhibition of initiation of mini-F plasmid replication in temperature-sensitive mafA mutants of Escherichia coli K-12

When temperature-sensitive mafA mutants of Escherichia coli K-12 carrying mini-F plasmid (pSC138) are transferred from 30 to 42 °C, plasmid DNA replication as determined by incorporation of [³H]thymidine into covalently closed circular (CCC) mini-F DNA or by DNA-DNA hybridization is inhibited markedly within 10 min. The results of extensive pulse-chase experiments suggest that the initiation rather than the chain elongation step of plasmid replication is affected under these conditions. The replication inhibition in the mutant is accompanied by appearance of a class of plasmid DNA with a buoyant density higher than that of CCC DNA observed in the wild type, and is followed by gradual inhibition of host cell growth. The inhibition of plasmid replication is reversible at least for 60 min under the conditions used, and the recovery at low temperature (30 °C) depends on the synthesis of untranslated RNA. These results taken together with other evidence suggest that the mafA mutations primarily affect the initial step(s) of F DNA replication, presumably at or before the synthesis of untranslated RNA.     

Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens

DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.     

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high‐energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA. However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La‐related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI‐Larp complex promotes the synthesis of a subset of nuclear‐encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI‐Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron‐transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI‐Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis.     

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled.     

Instability of inhibited replication forks in E. coli

Inhibiting the progress of replication forks in E. coli makes them susceptible to breakage. Broken replication forks are evidently reassembled by the RecBCD recombinational repair pathway. These findings explain a particular pattern of DNA degradation during inhibition of chromosomal replication, the role of recombination in the viability of mutants with displaced replication origin, and hyper-recombination observed in the Terminus of the E. coli chromosome in rnh mutants. Breakage and repair of inhibited replication forks could be the reason for the recombination-dependence of inducible stable DNA replication. A mechanism by which RecABCD-dependent recombination between very short inverted repeats may help E. coli to invert an operon, transcribed in the direction opposite to that of DNA replication, is discussed.     

Direct visualization of replication dynamics in early zebrafish embryos

We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2?-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.     

DNA gyrase activity regulates DnaA‐dependent replication initiation in Bacillus subtilis

In bacteria, initiation of DNA replication requires the DnaA protein. Regulation of DnaA association and activity at the origin of replication, oriC, is the predominant mechanism of replication initiation control. One key feature known to be generally important for replication is DNA topology. Although there have been some suggestions that topology may impact replication initiation, whether this mechanism regulates DnaA‐mediated replication initiation is unclear. We found that the essential topoisomerase, DNA gyrase, is required for both proper binding of DnaA to oriC as well as control of initiation frequency in Bacillus subtilis. Furthermore, we found that the regulatory activity of gyrase in initiation is specific to DnaA and oriC. Cells initiating replication from a DnaA‐independent origin, oriN, are largely resistant to gyrase inhibition by novobiocin, even at concentrations that compromise survival by up to four orders of magnitude in oriC cells. Furthermore, inhibition of gyrase does not impact initiation frequency in oriN cells. Additionally, deletion or overexpression of the DnaA regulator, YabA, significantly modulates sensitivity to gyrase inhibition, but only in oriC and not oriN cells. We propose that gyrase is a negative regulator of DnaA‐dependent replication initiation from oriC, and that this regulatory mechanism is required for cell survival.     

Attachment of DNA Loops to an Artificial Matrix Does Not Affect the Replication Origin Specificity in Early Development of Xenopus laevis

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.