Phylogenomics of Bryopsidales and the significance of genome duplication in multinucleate cells.

Centrioles get a helping hand from centrin

Multinucleated cells have been noted in pathophysiological states of the liver including infection with hepatitis B virus (HBV), the status of which is also closely associated with genomic instability in liver cancer. Here, we showed that hepatitis B virus X oncoprotein (HBx) expression in Chang cells results in a multinuclear phenotype and an abnormal number of centrosomes (n ≥ 3). Regulation of centrosome duplication in HBx-expressing ChangX-34 cells was defective and uncoupled from the cell cycle. HBx induced amplification of centrosomes, multipolar spindle formation, and chromosomal missegregation during mitosis and subsequently increased the generation of multinucleated cells and micronuclei formation. Treatment with PD98059, a mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2 inhibitor, significantly reduced the number of cells with hyperamplified centrosomes and decreased the multinucleated cells and micronuclei formation. Consistently, the phospho-ERK level during cell progression was substantially higher in ChangX-34 cells than that of Chang cells. In contrast, neither wortmannin, an inhibitor of phosphoinositide-3 kinase, nor SB203589, an inhibitor of p38 mitogen-activated protein kinase (MAPK), showed any effects. Introduction of Ras dominant-negative (D/N) and MEK2 D/N genes into ChangX-34 cells significantly alleviated centrosome amplification, whereas introduction of the PKC D/N and PKB D/N genes did not. Thus, our results demonstrate that the HBx induced centrosome hyperamplification and mitotic aberration by activation of the Ras-MEK-MAPK. Intervention of this signaling pathway could suppress the centrosome amplification as well as mitotic aberration. These findings may provide a possible mechanism by which HBx promotes phenotypic progression by predisposing chromosomal alteration in HBV-infected liver.

The exact duplication of the genetic material and its perfect segregation between the two products of cell division has to be tightly coordinated during the cell cycle. A number of genes involved in the orderly progression of events necessary for cell division have been identified in the yeast, Saccharomyces cerevisiae, by screening for mutants which under certain restrictive conditions (e.g. high temperature) stop to proliferate. Such cell division cycle (cdc) mutants arrest at certain stages of the cell cycle and show a “terminal phenotype” indicating which of the sequencial steps the mutated gene might be resposible for (for review see Pringle and Hartwell, 1981). In addition to genes and proteins absolutely essential for genome and cell duplication, the existence of functions bringing about valuable but not vital improvements for the precision of the process has to be assumed. Such genes will not be picked up by screening for conditionally lethal mutations.

Accurate genome duplication underlies genetic homeostasis. Metazoan Mdm2 binding protein (MTBP) forms a main regulatory platform for origin firing together with Treslin/TICRR and TopBP1 (Topoisomerase II binding protein 1 (TopBP1)–interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator). We report the first comprehensive analysis of MTBP and reveal conserved and metazoa-specific MTBP functions in replication. This suggests that metazoa have evolved specific molecular mechanisms to adapt replication principles conserved with yeast to the specific requirements of the more complex metazoan cells. We uncover one such metazoa-specific process: a new replication factor, cyclin-dependent kinase 8/19–cyclinC (Cdk8/19-cyclin C), binds to a central domain of MTBP. This interaction is required for complete genome duplication in human cells. In the absence of MTBP binding to Cdk8/19-cyclin C, cells enter mitosis with incompletely duplicated chromosomes, and subsequent chromosome segregation occurs inaccurately. Using remote homology searches, we identified MTBP as the metazoan orthologue of yeast synthetic lethal with Dpb11 7 (Sld7). This homology finally demonstrates that the set of yeast core factors sufficient for replication initiation in vitro is conserved in metazoa. MTBP and Sld7 contain two homologous domains that are present in no other protein, one each in the N and C termini. In MTBP the conserved termini flank the metazoa-specific Cdk8/19-cyclin C binding region and are required for normal origin firing in human cells. The N termini of MTBP and Sld7 share an essential origin firing function, the interaction with Treslin/TICRR or its yeast orthologue Sld3, respectively. The C termini may function as homodimerisation domains. Our characterisation of broadly conserved and metazoa-specific initiation processes sets the basis for further mechanistic dissection of replication initiation in vertebrates. It is a first step in understanding the distinctions of origin firing in higher eukaryotes.

Cdk2 has been viewed as a key cell cycle regulator that is essential for S phase progression. The recent discovery that Cdk2 is not required for cell proliferation in mice now shows that other factors must be able to replace Cdk2 in stimulating DNA replication. Experiments performed in Xenopus egg extracts identify the mitotic protein kinases Cdk1/Cyclin B and Cdk1/Cyclin A as likely candidates. These observations raise the intriguing possibility that Cdk1 normally participates in genome duplication in wild type cells.

In the yeast Saccharomyces cerevisiae DNA replication and spindle assembly can overlap. Therefore, signaling mechanisms modulate spindle dynamics in order to ensure correct timing of chromosome segregation relative to genome duplication, especially when replication is incomplete or the DNA becomes damaged. This review focuses on the molecular mechanisms that coordinate DNA replication and spindle dynamics, as well as on the role of spindle-dependent forces in DNA repair. Understanding the coupling between genome duplication and spindle function in yeast cells can provide important insights into similar processes operating in other eukaryotic organisms, including humans.

Genome stability is key for healthy cells in healthy organisms, and deregulated maintenance of genome integrity is a hallmark of aging and of age-associated diseases including cancer and neurodegeneration. To maintain a stable genome, genome surveillance and repair pathways are closely intertwined with cell cycle regulation and with DNA transactions that occur during transcription and DNA replication. Coordination of these processes across different time and length scales involves dynamic changes of chromatin topology, clustering of fragile genomic regions and repair factors into nuclear repair centers, mobilization of the nuclear cytoskeleton, and activation of cell cycle checkpoints. Here, we provide a general overview of cell cycle regulation and of the processes involved in genome duplication in human cells, followed by an introduction to replication stress and to the cellular responses elicited by perturbed DNA synthesis. We discuss fragile genomic regions that experience high levels of replication stress, with a particular focus on telomere fragility caused by replication stress at the ends of linear chromosomes. Using alternative lengthening of telomeres (ALT) in cancer cells and ALT-associated PML bodies (APBs) as examples of replication stress-associated clustered DNA damage, we discuss compartmentalization of DNA repair reactions and the role of protein properties implicated in phase separation. Finally, we highlight emerging connections between DNA repair and mechanobiology and discuss how biomolecular condensates, components of the nuclear cytoskeleton, and interfaces between membrane-bound organelles and membraneless macromolecular condensates may cooperate to coordinate genome maintenance in space and time.

Cancer stem‐like cells (CSCs) induce drug resistance and recurrence of tumors when they experience DNA replication stress. However, the mechanisms underlying DNA replication stress in CSCs and its compensation remain unclear. Here, we demonstrate that upregulated c‐Myc expression induces stronger DNA replication stress in patient‐derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini‐chromosome maintenance protein 10 (MCM10), a firing (activating) factor of DNA replication origins, to compensate for DNA replication stress in CSCs. MCM10 expression is upregulated in CSCs and is maintained by c‐Myc. c‐Myc‐dependent collisions between RNA transcription and DNA replication machinery may occur in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to these collisions to ensure the progression of replication. Moreover, patient‐derived breast CSCs were found to be dependent on MCM10 for their maintenance, even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. Further, MCM10 depletion decreased the growth of cancer cells, but not of normal cells. Therefore, MCM10 may robustly compensate for DNA replication stress and facilitate genome duplication in cancer cells in the S‐phase, which is more pronounced in CSCs. Overall, we provide a preclinical rationale to target the c‐Myc‐MCM10 axis for preventing drug resistance and recurrence of tumors.

BackgroundFanconi anemia (FA) is a rare disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer. Patients harboring X‐linked FANCB pathogenic variants usually present with severe congenital malformations resembling VACTERL syndrome with hydrocephalus.MethodsWe employed the diepoxybutane (DEB) test for FA diagnosis, arrayCGH for detection of duplication, targeted capture and next‐gen sequencing for defining the duplication breakpoint, PacBio sequencing of full‐length FANCB aberrant transcript, FANCD2 ubiquitination and foci formation assays for the evaluation of FANCB protein function by viral transduction of FANCB‐null cells with lentiviral FANCB WT and mutant expression constructs, and droplet digital PCR for quantitation of the duplication in the genomic DNA and cDNA.ResultsWe describe here an FA‐B patient with a mild phenotype. The DEB diagnostic test for FA revealed somatic mosaicism. We identified a 9154 bp intragenic duplication in FANCB, covering the first coding exon 3 and the flanking regions. A four bp homology (GTAG) present at both ends of the breakpoint is consistent with microhomology‐mediated duplication mechanism. The duplicated allele gives rise to an aberrant transcript containing exon 3 duplication, predicted to introduce a stop codon in FANCB protein (p.A319*). Duplication levels in the peripheral blood DNA declined from 93% to 7.9% in the span of eleven years. Moreover, the patient fibroblasts have shown 8% of wild‐type (WT) allele and his carrier mother showed higher than expected levels of WT allele (79% vs. 50%) in peripheral blood, suggesting that the duplication was highly unstable.ConclusionUnlike sequence point variants, intragenic duplications are difficult to precisely define, accurately quantify, and may be very unstable, challenging the proper diagnosis. The reversion of genomic duplication to the WT allele results in somatic mosaicism and may explain the relatively milder phenotype displayed by the FA‐B patient described here.

Many of the most basic biochemical mechanisms of DNA replication have been conserved from prokaryotes to eukaryotes, but the evolution of eukaryotic cells resulted in many changes in the logic of the cell cycle and in the mechanisms that regulate DNA replication. In a prokaryotic cell, such as Escherichia coli , DNA replication is generally initiated at a single essential locus in the circular chromosome (replicator). The timing and specificity of initiation are determined by the binding of a protein complex (initiator) to specific sequences in the replicator (Jacob et al. 1964). In a rapidly growing E. coli cell, initiation events occur at intervals significantly shorter than the time required to complete the synthesis of daughter chromosomes. Under these circumstances, DNA synthesis is essentially continuous throughout the cell cycle, and cells contain several partially completed chromosomes. In contrast, in eukaryotic cells, DNA replication occupies only part of the cell cycle and alternates with mitosis. Moreover, each chromosome is duplicated only once in each cell cycle, producing two complete daughter chromosomes. To ensure the timely completion of the replication of the large genomes of eukaryotes, initiation of DNA synthesis occurs at multiple chromosomal sites (Huberman and Riggs 1966). The number of such origins of DNA replication ranges from a few hundred in a yeast cell to tens of thousands in a human cell. Given the complexity of genome duplication in eukaryotic cells, it is not surprising that novel regulatory mechanisms have evolved to coordinate DNA replication with other events in the cell...

Chlamydia trachomatis infection has been suggested to induce host genome duplication and is linked to increased risks of cervical cancer. We describe here the mechanism by which Chlamydia causes a cleavage furrow defect that consistently results in the formation of multinucleated host cells, a phenomenon linked to tumorigenesis. Host signaling proteins essential for cleavage furrow initiation, ingression, and stabilization are displaced from one of the prospective furrowing cortices after Chlamydia infection. This protein displacement leads to the formation of a unique asymmetrical, unilateral cleavage furrow in infected human cells. The asymmetrical distribution of signaling proteins is caused by the physical presence of the Chlamydia inclusion at the cell equator. By using ingested latex beads, we demonstrate that the presence of a large vacuole at the cell equator is sufficient to cause furrow ingression failure and can lead to multinucleation. Interestingly, internalized latex beads of similar size do not localize to the cell equator as efficiently as Chlamydia inclusions; moreover, inhibition of bacterial protein synthesis with antibiotic reduces the frequency at which Chlamydia localizes to the cell equator. Together, these results suggest that Chlamydia effectors are involved in strategic positioning of the inclusion during cell division.

Osteopontin (OPN) is a secreted phosphoprotein involved in cellular proliferation and associated with tumor progression. Although an intracellular form of OPN has been described, its function remains unknown. In this study, a novel nuclear location for intracellular OPN and a correlation with cell division were demonstrated. OPN distinctly localized to the nucleus in a subset of transiently transfected human embryonic kidney 293 cells. Immunoblotting confirmed the nuclear location of native OPN, and results from immunofluorescence studies suggested an association between nuclear OPN and cell cycle progression. Flow cytometry revealed that nuclear and cellular OPN content rose significantly during the S and G(2)/M phases, respectively. Treatment of cells with the DNA polymerase inhibitor aphidicolin prevented cell cycling and greatly reduced cellular OPN content. The intracellular location of OPN coincided with polo-like kinase-1 (Plk-1), a member of the polo-like kinase family, which, in part through their regulation of centrosome-related events, are integral to successful cellular mitosis. OPN and Plk-1 were coimmunoprecipitated from nuclear, but not cystoslic, extracts, demonstrating an interaction that is limited to the nucleus, presumably during mitosis. Deletion of the COOH terminus of OPN militated against nuclear localization and Plk-1 interaction. Elevated expression of OPN was also associated with an increase in the number of multinucleate 293 cells, whereas transfection of the COOH-terminal-deleted OPN decreased the percentage of multinucleate cells below basal levels. These findings implicate intranuclear OPN as a participant in the process of cell duplication.

Chapter 48 Flow Cytometric Analysis of Plant Genomes

The cell energy fraction that powered maintenance and expression of genes encoding pro-phage elements, pta-ack cluster, early sporulation, sugar ABC transporter periplasmic proteins, 6-phosphofructokinase, pyruvate kinase, and fructose-1,6-disphosphatase in acetogen Clostridium sp. MT871 was re-directed to power synthetic operon encoding isobutanol biosynthesis at the expense of these genes achieved via their elimination. Genome tailoring decreased cell duplication time by 7.0 ± 0.1 min (p < 0.05) compared to the parental strain, with intact genome and cell duplication time of 68 ± 1 min (p < 0.05). Clostridium sp. MT871 with tailored genome was UVC-mutated to withstand 6.1 % isobutanol in fermentation broth to prevent product inhibition in an engineered commercial biocatalyst producing 5 % (674.5 mM) isobutanol during two-step continuous fermentation of CO2/H2 gas blend. Biocatalyst Clostridium sp. MT871RG- 11IBR6 was engineered to express six copies of synthetic operon comprising optimized synthetic format dehydrogenase, pyruvate formate lyase, acetolactate synthase, acetohydroxyacid reductoisomerase, 2,3-dihydroxy-isovalerate dehydratase, branched-chain alpha-ketoacid decarboxylase gene, aldehyde dehydrogenase, and alcohol dehydrogenase, regaining cell duplication time of 68 ± 1 min (p < 0.05) for the parental strain. This is the first report on isobutanol production by an engineered acetogen biocatalyst suitable for commercial manufacturing of this chemical/fuel using continuous fermentation of CO2/H2 blend thus contributing to the reversal of global warming.

Faithful chromosome segregation of 8 duplicated haploid genomes into 8 daughter gametes is essential for male gametogenesis and mosquito transmission of Plasmodium. Plasmodium undergoes endomitosis in this multinucleated cell division, which is highly reliant on proper spindle-kinetochore attachment. However, the mechanisms underlying the spindle-kinetochore attachment remain elusive. End-binding proteins (EBs) are conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. Here, we report that the Plasmodium EB1 is an orthologue distinct from the canonical eukaryotic EB1. Both in vitro and in vivo assays reveal that the Plasmodium EB1 losses MT plus-end tracking but possesses MT-lattice affinity. This MT-binding feature of Plasmodium EB1 is contributed by both CH domain and linker region. EB1-deficient parasites produce male gametocytes that develop to the anucleated male gametes, leading to defective mosquito transmission. EB1 is localized at the nucleoplasm of male gametocytes. During the gametogenesis, EB1 decorates the full-length of spindle MTs and regulates spindle structure. The kinetochores attach to spindle MTs laterally throughout endomitosis and this attachment is EB1-dependent. Consequently, impaired spindle-kinetochore attachment is observed in EB1-deficient parasites. These results indicate that a parasite-specific EB1 with MT-lattice binding affinity fulfills the spindle-kinetochore lateral attachment in male gametogenesis.