Multiple Rounds Of DNA Replication Research Articles

Genome-wide binding sites of Plasmodium falciparum mini chromosome maintenance protein MCM6 show new insights into parasite DNA replication.

Multiple rounds of DNA replication take place in various stages of the life cycle in the human malaria parasite Plasmodium falciparum. Previous bioinformatics analysis has shown the presence of putative Autonomously Replicating Sequence (ARS) like sequences in the Plasmodium genome. However, the actual sites and frequency of replication origins in the P. falciparum genome based on experimental data still remain elusive. Minichromosome maintenance (MCM) proteins are recruited by the Origin recognition complex (ORC) to the origins of replication in eukaryotes including P. falciparum. We used PfMCM6 for chromatin immunoprecipitation followed by sequencing (ChIP-seq) in the quest for identification of putative replication origins in the parasite. PfMCM6 DNA binding sites annotation revealed high enrichment at exon regions. This is contrary to higher eukaryotes that show an inclination of origin sites towards transcriptional start sites. ChIP-seq results were further validated by ChIP-qPCR results as well as nascent strand abundance assay at the selected PfMCM6 enriched sites that also showed preferential binding of PfORC1 suggesting potential of these sites as origin sites. Further, PfMCM6 ChIP-seq data showed a positive correlation with previously published histone H4K8Ac genome-wide binding sites but not with H3K9Ac sites suggesting epigenetic control of replication initiation sites in the parasites. Overall, our data show the genome-wide distribution of PfMCM6 binding sites with their potential as replication origins in this deadly human pathogen that not only broadens our knowledge of parasite DNA replication and its unique biology, it may help to find new avenues for intervention processes.

Myc promotes polyploidy in murine trophoblast cells and suppresses senescence.

The placenta is essential for reproductive success. The murine placenta includes polyploid giant cells that are crucial for its function. Polyploidy occurs broadly in nature but its regulators and significance in the placenta are unknown. We have discovered that many murine placental cell types are polyploid and have identified factors that license polyploidy using single-cell RNA sequencing. Myc is a key regulator of polyploidy and placental development, and is required for multiple rounds of DNA replication, likely via endocycles, in trophoblast giant cells. Furthermore, MYC supports the expression of DNA replication and nucleotide biosynthesis genes along with ribosomal RNA. Increased DNA damage and senescence occur in trophoblast giant cells without Myc, accompanied by senescence in the neighboring maternal decidua. These data reveal Myc is essential for polyploidy to support normal placental development, thereby preventing premature senescence. Our study, combined with available literature, suggests that Myc is an evolutionarily conserved regulator of polyploidy.

A genome-wide map of DNA replication at single-molecule resolution in the malaria parasite Plasmodium falciparum.

The malaria parasite Plasmodium falciparum replicates via schizogony: an unusual type of cell cycle involving asynchronous replication of multiple nuclei within the same cytoplasm. Here, we present the first comprehensive study of DNA replication origin specification and activation during Plasmodium schizogony. Potential replication origins were abundant, with ORC1-binding sites detected every ∼800 bp. In this extremely A/T-biased genome, the sites were biased towards areas of higher G/C content, and contained no specific sequence motif. Origin activation was then measured at single-molecule resolution using newly developed DNAscent technology: a powerful method of detecting replication fork movement via base analogues in DNA sequenced on the Oxford Nanopore platform. Unusually, origins were preferentially activated in areas of low transcriptional activity, and replication forks also moved fastest through lowly transcribed genes. This contrasts with the way that origin activation is organised in other systems, such as human cells, and suggests that P. falciparum has evolved its S-phase specifically to minimise conflicts between transcription and origin firing. This may be particularly important to maximise the efficiency and accuracy of schizogony, with its multiple rounds of DNA replication and its absence of canonical cell-cycle checkpoints.

Gametogenesis in Plasmodium: Delving Deeper to Connect the Dots.

In the coming decades, eliminating malaria is the foremost goal of many tropical countries. Transmission control, along with an accurate and timely diagnosis of malaria, effective treatment and prevention are the different aspects that need to be met synchronously to accomplish the goal. The current review is focused on one of these aspects i.e., transmission control, by looking deeper into the event called gametogenesis. In the Plasmodium life cycle, gametocytes are the first life forms of the sexual phase. The transmission of the parasite and the disease is critically dependent on the number, viability and sex ratio of mature gametocytes and their further development inside mosquito vectors. Gametogenesis, the process of conversion of gametocytes into viable gametes, takes place inside the mosquito midgut, and is a tightly regulated event with fast and multiple rounds of DNA replication and diverse cellular changes going on within a short period. Interrupting the gametocyte-gamete transition is ought to restrict the successful transmission and progression of the disease and hence an area worth exploring for designing transmission-blocking strategies. This review summarizes an in-depth and up-to-date understanding of the biochemical and physiological mechanism of gametogenesis in Plasmodium, which could be targeted to control parasite and malaria transmission. This review also raises certain key questions regarding gametogenesis biology in Plasmodium and brings out gaps that still accompany in understanding the spectacular process of gametogenesis.

Characterization of Growth and Cell Cycle Events Affected by Light Intensity in the Green Alga Parachlorella kessleri: A New Model for Cell Cycle Research.

Multiple fission is a cell cycle variation leading to the production of more than two daughter cells. Here, we used synchronized cultures of the chlorococcal green alga Parachlorella kessleri to study its growth and pattern of cell division under varying light intensities. The time courses of DNA replication, nuclear and cellular division, cell size, total RNA, protein content, dry matter and accumulation of starch were observed at incident light intensities of 110, 250 and 500 µmol photons m−2s−1. Furthermore, we studied the effect of deuterated water on Parachlorella kessleri growth and division, to mimic the effect of stress. We describe a novel multiple fission cell cycle pattern characterized by multiple rounds of DNA replication leading to cell polyploidization. Once completed, multiple nuclear divisions were performed with each of them, immediately followed by protoplast fission, terminated by the formation of daughter cells. The multiple fission cell cycle was represented by several consecutive doublings of growth parameters, each leading to the start of a reproductive sequence. The number of growth doublings increased with increasing light intensity and led to division into more daughter cells. This study establishes the baseline for cell cycle research at the molecular level as well as for potential biotechnological applications, particularly directed synthesis of (deuterated) starch and/or neutral lipids as carbon and energy reserves.

Active DNA demethylation-The epigenetic gatekeeper of development, immunity, and cancer.

DNA methylation is a critical process in the regulation of gene expression with dramatic effects in development and continually expanding roles in oncogenesis. 5-Methylcytosine was once considered to be an inherited and stably repressive epigenetic mark, which can be only removed by passive dilution during multiple rounds of DNA replication. However, in the past two decades, physiologically controlled DNA demethylation and deamination processes have been identified, thereby revealing the function of cytosine methylation as a highly regulated and complex state-not simply a static, inherited signature or binary on-off switch. Alongside these fundamental discoveries, clinical studies over the past decade have revealed the dramatic consequences of aberrant DNA demethylation. In this review we discuss DNA demethylation and deamination in the context of 5-methylcytosine as critical processes for physiological and physiopathological transitions within three states-development, immune maturation, and oncogenic transformation; and we describe the expanding role of DNA demethylating drugs as therapeutic agents in cancer.

Persistent DNA Damage and Senescence in the Placenta Impacts Developmental Outcomes of Embryos.

Cohesin is an evolutionarily conserved chromosome-associated protein complex essential for chromosome segregation, gene expression, and repair of DNA damage. Mutations that affect this complex cause the human developmental disorder Cornelia de Lange syndrome (CdLS), thought to arise from defective embryonic transcription. We establish a significant role for placental defects in the development of CdLS mouse embryos (Nipbl and Hdac8). Placenta is a naturally senescent tissue; we demonstrate that persistent DNA damage potentiates senescence and activates cytokine signaling. Mutant embryo developmental outcomes are significantly improved in the context of a wild-type placenta or by genetically restricting cytokine signaling. Our study highlights that cohesin is required for maintaining ploidy and the repair of spontaneous DNA damage in placental cells, suggesting that genotoxic stress and ensuing placental senescence and cytokine production could represent a broad theme in embryo health and viability.

Centromere and its associated proteins-what we know about them in Plasmodium falciparum.

The complex life cycle of intracellular parasitic protozoans entails multiple rounds of DNA replication and mitosis followed by cytokinesis to release daughter parasites. To gain insights into mitotic events it is imperative to identify the biomarkers that constitute the chromosome segregation machinery in the parasite. Chromosomal loci called centromeres and their associated proteins play an essential role in accurate chromosome segregation. Although new information on the centromere-kinetochore proteins has been added to the existing pool of knowledge, a paucity of biomarkers for nuclear division prevents a global view of chromosome segregation mechanism in the malaria parasite. In Plasmodium falciparum, except CENH3 and CENP-C homologues, other centromere associated proteins responsible for centromere functions and kinetochore assembly are not known. The focus of this review is to summarize the current understanding on the centromere organization and its associated proteins in eukaryotes with the emerging information in P. falciparum. © 2018 IUBMB Life, 70(8):732-742, 2018.

Chromosomal instability during neurogenesis in Huntington's disease.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by expansion of CAG repeats in the Huntingtin gene (HTT). Neither its pathogenic mechanisms nor the normal functions of HTT are well understood. To model HD in humans, we engineered a genetic allelic series of isogenic human embryonic stem cell (hESC) lines with graded increases in CAG repeat length. Neural differentiation of these lines unveiled a novel developmental HD phenotype: the appearance of giant multinucleated telencephalic neurons at an abundance directly proportional to CAG repeat length, generated by a chromosomal instability and failed cytokinesis over multiple rounds of DNA replication. We conclude that disrupted neurogenesis during development is an important, unrecognized aspect of HD pathogenesis. To address the function of normal HTT protein we generated HTT+/- and HTT-/- lines. Surprisingly, the same phenotype emerged in HTT-/- but not HTT+/- lines. We conclude that HD is a developmental disorder characterized by chromosomal instability that impairs neurogenesis, and that HD represents a genetic dominant-negative loss of function, contrary to the prevalent gain-of-toxic-function hypothesis. The consequences of developmental alterations should be considered as a new target for HD therapies.

Identification and characterization of ARS-like sequences as putative origin(s) of replication in human malaria parasite Plasmodium falciparum.

DNA replication is a fundamental process in genome maintenance, and initiates from several genomic sites (origins) in eukaryotes. In Saccharomyces cerevisiae, conserved sequences known as autonomously replicating sequences (ARSs) provide a landing pad for the origin recognition complex (ORC), leading to replication initiation. Although origins from higher eukaryotes share some common sequence features, the definitive genomic organization of these sites remains elusive. The human malaria parasite Plasmodium falciparum undergoes multiple rounds of DNA replication; therefore, control of initiation events is crucial to ensure proper replication. However, the sites of DNA replication initiation and the mechanism by which replication is initiated are poorly understood. Here, we have identified and characterized putative origins in P. falciparum by bioinformatics analyses and experimental approaches. An autocorrelation measure method was initially used to search for regions with marked fluctuation (dips) in the chromosome, which we hypothesized might contain potential origins. Indeed, S. cerevisiae ARS consensus sequences were found in dip regions. Several of these P. falciparum sequences were validated with chromatin immunoprecipitation-quantitative PCR, nascent strand abundance and a plasmid stability assay. Subsequently, the same sequences were used in yeast to confirm their potential as origins in vivo. Our results identify the presence of functional ARSs in P. falciparum and provide meaningful insights into replication origins in these deadly parasites. These data could be useful in designing transgenic vectors with improved stability for transfection in P. falciparum.

Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony.

Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood-stage of infection1. DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly2,3. However, the control mechanisms for this divergent mode of replication are unknown. Here we show that the Plasmodium-specific kinase PfCRK4 is a key cell cycle regulator that orchestrates the multiple rounds of DNA replication throughout schizogony in P. falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in origin of replication firing. PfCRK4 was required for the initial and subsequent rounds of DNA replication during schizogony, and in addition was essential for development in the mosquito vector. Our results identified an essential S phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic blood-stage of malaria infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.

Global Analysis of Protein Expression and Phosphorylation of Three Stages of Plasmodium falciparum Intraerythrocytic Development

During asexual intraerythrocytic development, Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the Plasmodium asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase-substrate pairs operative in various signaling networks in the parasite.

Light-Dependent Regulation ofDEL1Is Determined by the Antagonistic Action of E2Fb and E2Fc

Endoreduplication represents a variation on the cell cycle in which multiple rounds of DNA replication occur without subsequent chromosome separation and cytokinesis, thereby increasing the cellular DNA content. It is known that the DNA ploidy level of cells is controlled by external stimuli such as light; however, limited knowledge is available on how environmental signals regulate the endoreduplication cycle at the molecular level. Previously, we had demonstrated that the conversion from a mitotic cell cycle into an endoreduplication cycle is controlled by the atypical E2F transcription factor, DP-E2F-LIKE1 (DEL1), that represses the endocycle onset. Here, the Arabidopsis (Arabidopsis thaliana) DEL1 gene was identified as a transcriptional target of the classical E2Fb and E2Fc transcription factors that antagonistically control its transcript levels through competition for a single E2F cis-acting binding site. In accordance with the reported opposite effects of light on the protein levels of E2Fb and E2Fc, DEL1 transcription depended on the light regime. Strikingly, modified DEL1 expression levels uncoupled the link between light and endoreduplication in hypocotyls, implying that DEL1 acts as a regulatory connection between endocycle control and the photomorphogenic response.

Caspase-3–Dependent Mitotic Checkpoint Inactivation by the Small-Molecule Inducers of Mitotic Slippage SU6656 and Geraldol

Microtubule-targeting cancer drugs such as paclitaxel block cell-cycle progression at mitosis by prolonged activation of the mitotic checkpoint. Cells can spontaneously escape mitotic arrest and enter interphase without chromosome segregation by a process termed mitotic slippage that involves the degradation of cyclin B1 without mitotic checkpoint inactivation. Inducing mitotic slippage with chemicals causes cells to die after multiple rounds of DNA replication without cell division, which may enhance the antitumor activity of microtubule-targeting drugs. Here, we explore pathways leading to mitotic slippage by using SU6656 and geraldol, two recently identified chemical inducers of mitotic slippage. Mitotic slippage induced by SU6656 or geraldol was blocked by the proteasome inhibitor MG-132 and involved proteasome-dependent degradation of cyclin B1 and the mitotic checkpoint proteins budding uninhibited by benzimidazole related 1 (BubR1) and cell division cycle 20 (Cdc20) in T98G cells. Mitotic slippage and the degradation of BubR1 and Cdc20 were also inhibited by the caspase-3 and -7 inhibitor DEVD-CHO. MCF-7 cells lacking caspase-3 expression could not degrade BubR1 or undergo mitotic slippage in response to SU6656 or geraldol. Introduction of caspase-3 completely restored the ability of MCF-7 cells to degrade BubR1 and undergo mitotic slippage. However, lack of expression of caspase-3 did not affect cell death after exposure to paclitaxel, with or without mitotic slippage induction. The requirement for caspase-3 for chemically induced mitotic slippage reveals a new mechanism for mitotic exit and a link between mitosis and apoptosis that has implications for the outcome of cancer chemotherapy.

SP600125 suppresses Cdk1 and induces endoreplication directly from G2 phase, independent of JNK inhibition

Cell cycle controls ensure that DNA replication (S phase) follows mitosis resulting in two precise copies of the genome. A failure of the control mechanisms can result in multiple rounds of DNA replication without cell division. In endoreplication, cells with replicated genomes bypass mitosis, then replicate their DNA again, resulting in polyploidy. Endoreplication from G2 phase lacks all hallmarks of mitosis. Using synchronized cells, we show that the c-Jun N-terminal kinase (JNK) inhibitor, SP600125, prevents the entry of cells into mitosis and leads to endoreplication of DNA from G2 phase. We show that cells proceed from G2 phase to replicate their DNA in the absence of mitosis. This effect of SP600125 is independent of its suppression of JNK activity. Instead, the inhibitory effect of SP600125 on mitotic entry predominantly occurs upstream of Aurora A kinase and Polo-like kinase 1, resulting in a failure to remove the inhibitory phosphorylation of Cdk1. Importantly, our results directly show that the inhibition of Cdk1 activity and the persistence of Cdk2 activity in G2 cells induces endoreplication without mitosis. Furthermore, endoreplication from G2 phase is independent of p53 control.

The anaphase-promoting complex/cyclosome (APC/C) is required for rereplication control in endoreplication cycles

Endoreplicating cells undergo multiple rounds of DNA replication leading to polyploidy or polyteny. Oscillation of Cyclin E (CycE)-dependent kinase activity is the main driving force in Drosophila endocycles. High levels of CycE-Cdk2 activity trigger S phase, while down-regulation of CycE-Cdk2 activity is crucial to allow licensing of replication origins. In mitotic cells relicensing in S phase is prevented by Geminin. Here we show that Geminin protein oscillates in endoreplicating salivary glands of Drosophila. Geminin levels are high in S phase, but drop once DNA replication has been completed. DNA licensing is coupled to mitosis through the action of the anaphase-promoting complex/cyclosome (APC/C). We demonstrate that, even though endoreplicating cells never enter mitosis, APC/C activity is required in endoreplicating cells to mediate Geminin oscillation. Down-regulation of APC/C activity results in stabilization of Geminin protein and blocks endocycle progression. Geminin is only abundant in cells with high CycE-Cdk2 activity, suggesting that APC/C-Fzr activity is periodically inhibited by CycE-Cdk2, to prevent relicensing in S-phase cells.

Cdt1 and geminin: role during cell cycle progression and DNA damage in higher eukaryotes

DNA replication in eukaryotic cells must be strictly regulated to ensure that the entire genome is duplicated only once in each cell cycle. For this purpose, the initiation of DNA replication is controlled by the "licensing" reaction, which is established by the formation of a pre-replicative complex (pre-RC) with the sequential assembly of the origin recognition complex (ORC), Cdc6, Cdt1 and Mcm2-7 onto origin regions. Among these, Cdt1 is likely the most important target for regulating licensing in higher eukaryotic cells, since illegitimate accumulation of Cdt1 causes multiple rounds of DNA replication without an intervening mitosis. Cdt1 is regulated over the course of the cell cycle mainly by the controlled expression of an inhibitor protein, geminin, and the level of Cdt1 periodically fluctuates due to ubiquitination and proteolysis. While the expression of geminin from S phase to metaphase of mitosis prevents licensing, Cdt1 accumulates from M to G1 phases and is degraded at the onset of S phase. Furthermore, Cdt1 is also proteolyzed in G1 phase in response to DNA damage, presumably providing a new checkpoint control.

Reconstitution ofSaccharomyces cerevisiaeprereplicative complex assemblyin vitro

The assembly of the prereplicative complex (pre-RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre-RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre-RC assembly was reconstituted in vitro using ARS1 DNA and purified origin-recognition complex (ORC), Cdc6p and Cdt1p-Mcm2-7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2-7p on to ARS1 DNA. When Mcm2-7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co-ordinately regulated during pre-RC assembly. In extracts from sid2-21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2-7p do not. However, Mcm2-7p does bind in the presence of exogenous Cdt1p or Cdt1p-Mcm2-7p complex. Cdt1p-Mcm2-7p complex, which was purified from G1-, early S or G2/M-arrested cells, exhibits structure-specific DNA binding, interacting only with bubble- or Y-shape-DNA, but the biological significance of this observation is not yet known.

Progeny of germ line knockouts of ASI2, a gene encoding a putative signal transduction receptor in Tetrahymena thermophila, fail to make the transition from sexual reproduction to vegetative growth

The ciliated protozoan Tetrahymena has two nuclei: a germ line micronucleus and a somatic macronucleus. The transcriptionally active macronucleus has about 50 copies of each chromosome. At sexual reproduction (conjugation), the parental macronucleus is degraded and new macronucleus develops from a mitotic product of the zygotic micronucleus. Development of the macronucleus involves massive genome remodeling, including deletion of about 6000 specific internal eliminated sequences (IES) and multiple rounds of DNA replication. A gene encoding a putative signal transduction receptor, ASI2, (anlagen stage induced 2) is up-regulated during development of the new macronuclei (anlagen). Macronuclear ASI2 is nonessential for vegetative growth. Homozygous ASI2 germ line knockout cells with wild type parental macronuclei proceed through mating but arrest at late macronuclear anlagen development and die before the first post-conjugation fission. IES elimination occurs in these cells. Two rounds of postzygotic DNA replication occur normally in progeny of ASI2 germ line knockouts, but endoreduplication of the macronuclear genome is arrested. The germ line ASI2 null phenotype is rescued in a mating of a knockout strain with wild type cells.

High levels of Cdc7 and Dbf4 proteins can arrest cell-cycle progression

Cdc7-Dbf4 serine/threonine kinase is essential for initiation of DNA replication. It was previously found that overexpression of certain replication proteins such as Cdc6 and Cdt1 in fission yeast resulted in multiple rounds of DNA replication in the absence of mitosis. Since this phenomenon is dependent upon the presence of wild-type Cdc7/Hsk1, we hypothesized that high levels of Cdc7 and/or Dbf4 could also cause multiple rounds of DNA replication, or could facilitate entry into S phase. To test this hypothesis, we transiently overexpressed hamster Cdc7, Dbf4 or both in CHO cells. Direct observations of individual cells by fluorescence microscopy and flow cytometric analysis on cell populations suggest that overexpression of Cdc7 and/or Dbf4 does not result in multiple rounds of DNA replication or facilitating entry into S phase. In contrast, moderately increased levels of Dbf4, but not Cdc7, cause cell-cycle arrest in G2/M. This G2/M arrest coincides with hyperphosphorylation of Cdc2/Cdk1 at Tyr-15, raising the possibility that high levels of Dbf4 may activate a G2/M cell-cycle checkpoint. Further increase in Cdc7 and/or Dbf4 by 2–4 fold can arrest cells in G1 and significantly slow down S-phase progression for the cells already in S phase.