Thus, changing from an S-phase state to a G2-phase state requires release of a brake, rather than the generation of a new trigger Fig. Mitosis is sensitive to persisting replication intermediates that can prevent sister chromatid separation. Hence, persisting replication intermediates are cut by structure-specific nucleases to promote faithful sister chromatid disjunction Chan and West, ; Naim et al. While cleavage of a few remaining replication intermediates during mitosis might be beneficial, such activities in S-phase would cause genome-wide havoc.
Different mechanisms have evolved to restrict endonucleases to mitosis. Reinstating double-strand break repair during mitosis is highly toxic and causes telomere fusions Orthwein et al. Replication stress can be caused by many factors, including deregulation of CDK activity by oncogenes such as cyclin E and Cdc25A Beck et al. Interestingly, forced mitotic entry by WEE1 inhibition requires the structure-specific DNA nuclease Mus81, suggesting that stalled forks need to be processed to allow premature mitotic entry Duda et al.
The quantitative threshold model for cell cycle progression, originally introduced by Stern and Nurse to explain that a single cyclin-CDK can drive the fission yeast cell cycle, has for decades stood the test of time Coudreuse and Nurse, ; Swaffer et al. The quantitative model focuses on the amount of CDK activity rather than the identity of different cyclin-CDK complexes.
In this model, S-phase requires relatively low cyclin-CDK activity, whereas mitosis only occurs above a much higher threshold of cyclin-CDK activity. Although likely complemented with different specificities of various cyclin-CDK complexes in other eukaryotes, this model explains why DNA replication commences before cell division Hochegger et al.
However, it raises the question of how the cell cycle control system is wired to ensure that cyclin-CDK activity does not rise too quickly. As discussed earlier, DNA replication is sensitive to high CDK activity and needs to be completed before cell division. Increasing cyclin-CDK activity can in principle be accomplished by triggering additional sets of engines that push cyclin-CDK activity to higher levels.
Accordingly, the road to mitosis is sometimes envisioned as a mountain. Approaching mitosis, the slope of the mountain is very steep, reflecting the high levels of CDK activity required for cell division Fig. The increasing CDK activity observed in cycling cells is primarily due to the presence of multiple positive feedback loops Lindqvist et al. These feedback loops ensure that once CDK activation is initiated, it will autonomously continue to increase in an exponential fashion, similar to a ball rolling down a mountain picking up speed without the need for extra engines.
As discussed, progressing from an S-phase state to a G2-phase state requires the release of a brake DNA replication , rather than the generation of a new trigger. Thus, mitotic entry per se does not require an extra engine or trigger in G2-phase, but instead might involve a single trigger in G1-phase and a set of counteracting brakes. These molecular brakes ensure that CDK activation occurs in a stepwise manner and on par with the processes required for faithful cell division.
For instance, CDK-dependent FOXM1 phosphorylation is initiated early but plateaus during S-phase, which postpones full activation of the promitotic transcriptional program until after genome duplication Saldivar et al. We believe recent discoveries fit a model in which the road to mitotic entry, instead of a steep climb, resembles a well-monitored descent Fig.
We here propose a model in which the descent is controlled by three brake modules that collectively determine CDK activity output and thus the timing of mitosis Fig.
Instead of focusing solely on the source and amount of CDK activity, we propose that it is more informative to think of the cell cycle in terms of an energy landscape, as is frequently done for chemical reactions, and for cell biology has been made popular by the cellular route to differentiation Takahashi, ; Waddington, Because the signaling landscape in which a given CDK activity acts is critical to its outcome, we propose that the kinetics of the cell cycle relies on the condition of the road and strength of the brakes rather than the power of the engine.
While all three brakes regulate CDK activity, they have different effectors and act at different stages of the cell cycle. In unchallenged conditions, the release of each brake corresponds to a cell cycle transition. Linking spiraling CDK activation to dual negative feedback loops ensures full commitment into S-phase Fig.
In other words, the cell pushes the gas and brake at the same time, yet the brake is inherently transient. The M-entry brake is a composite brake module that acts both on CDK itself and on its substrates.
However, the factors that enforce the cell cycle delay i. The timing of cell cycle transitions can be explained by the sequential release of three essential brake signals that counteract CDK activation Fig. Thus far we have mainly focused on the intrinsic signaling circuits that determine cell cycle progression, but cell proliferation is highly sensitive to external cues.
A multitude of developmental or experimental conditions can slow down or arrest the cell cycle Edgar and Lehner, ; Elledge, ; Kipreos and van den Heuvel, Examples of effectors of DNA damage checkpoints are ATM and the pp21 axis, which are critical for maintaining genome integrity but are not required for cell cycle progression per se Bartkova et al.
In G2-phase, this depends on the self-amplifying properties of promitotic signaling, which eventually overcomes checkpoint signaling Jaiswal et al.
The G2 DNA damage checkpoint therefore delays rather than blocks mitosis, arguing that also DNA damage checkpoints can function as brakes rather than strict checkpoint barriers. Examples of brake effectors for each class are depicted in Fig. The process of DNA replication can modulate the timing and amplitude of both types of brakes. The activities capable of copying a living cell have intrigued scientists for decades, and through the years many fundamental concepts of the cell cycle have been exposed Baserga, ; Dephoure et al.
One key feature of cycling cells is the distinct surge of DNA incorporation in S-phase, in which the cell duplicates its genome before cell division. It is now clear that DNA replication is not just an output of the cell cycle, but in fact feeds back into the signaling networks controlling mitotic entry. Linking DNA replication to the mitotic entry network dismisses the need for separate triggers while allowing temporal separation. We propose a cell cycle model based on a single trigger, which together with a set of three molecular brakes generates distinct waves of protein activities.
This brake model can explain gradual cyclin-CDK activation and distinct phase transitions, as well as commitment to complete the cell cycle once initiated. Many aspects of this model require further study. Are there situations in which additional triggers are needed? Plasmodium encodes the basic replicative machinery that is found in all eukaryotes, including DNA polymerases [ 21 , 22 ], proliferating cell nuclear antigen PCNA [ 23 , 24 ] and minichromosome maintenance proteins MCMs [ 25 , 26 ].
The remaining members of the complex are either absent or lack sufficient homology with characterised members to allow clear identification, although in P. Cdc6 is required for the recruitment of Cdt1 and the loading of MCM proteins to date only a putative Cdt1-like gene has been identified in Plasmodium. Sequence analysis has failed to identify a clear homologue of Cdc45 in Plasmodium , while the members of the GINS complex have been putatively identified, based on low sequence homology, but it remains to be determined whether they are functional.
It recognises a conserved consensus sequence in the yeast Saccharomyces cerevisiae , but in other eukaryotes there is no consensus sequence and the preferred composition of DNA bound by ORC varies from organism to organism [ 29 ]. Putative ORC-binding sequences in P.
The rate of replication was not constant, but decreased as the cells neared completion of schizogony, coinciding with a reduction in the mean distance between individual origins. Interestingly, this is the opposite of the pattern seen in human cells, where replication speeds up and origins become more widely spaced as S phase proceeds.
Development of techniques to examine the replication of the Plasmodium genome. Origins in S. Following reversible DNA-protein cross linking, the genome is fragmented and the proteins of interest are purified along with the associated DNA fragments, which are then sequenced.
This may include origins that would never be activated, and may miss those where the protein complex has dissociated from the chromosome.
Parasites expressing viral thymidine kinase can incorporate the synthetic nucleosides IdU red and CldU green which can be visualised in individual nuclei or on combed DNA fibres, allowing the calculation of inter-origin distances and replication rates. During erythrocytic schizogony the first replication and possible nuclear division requires 4—6 hours [ 13 ]. This, however, is not the maximal rate of replication for the parasite. After ingestion by a mosquito, male gametocytes undergo a 3-fold replication of their genome in less than 15 minutes, resulting in the production of 8 motile microgametes with 1N genome content [ 4 , 5 ].
Female gametocytes also mature and exit their host cells upon entering a mosquito but no replication or cell division occurs. In the model rodent malaria species P. Such extreme speed is unprecedented in eukaryotic gametogenesis, and may reflect strong pressure to complete the sexual cycle and exit the midgut before the parasite cells are digested along with the blood meal.
If the replication speed remains the same as it is in erythrocytic schizogony, then almost all of the suggested ORC-binding sites [ 30 ] must be used as origins simultaneously. Precedents do exist for such extremely flexible origin usage: in the earliest replications of Xenopus embryos, origins occur every 5—15 kb, spacing out only after the mid-blastula transition [ 33 ].
Plasmodium genome replication may be under similarly flexible control, although nothing is yet known about how this might be differentially enforced in gametogenesis, sporogony, hepatic and erythrocytic schizogony. As described above, Plasmodium undergoes multiple unconventional cell cycles, in a variety of host cell types and for varying durations. Although the genomic revolution for Plasmodium has permitted some investigation of these regulators, our understanding at present is patchy and incomplete.
In eukaryotic cells, cell cycle progression is governed by cyclins and cyclin-dependent protein kinases CDKs , along with other proteins such the anaphase promoting complex APC , which promotes waves of cyclin degradation.
The interplay between these regulatory and catalytic components and their timely upregulation, inhibition and degradation prompts sequential progression through G1, S, G2 and M phases [ 34 ] Fig. The peculiarities of Plasmodium schizogony begin with the lack of a G2 phase as the syncytial nuclei appear to alternate asynchronously between S and M phases prior to the orchestrated event of cytokinesis [ 35 ] Fig.
This raises questions about whether control of replicative cycles through diffusible cytoplasmic factors is feasible [ 2 , 12 ]. Illustration of cell cycle phases in Plasmodium erythrocytic schizogony a and Plasmodium male gametogenesis b. The predicted involvement of cyclins, CDKs and other kinases is shown at each phase. Placement of such components is only loosely chronological since most details are unknown. Crks or CDKs predicted to be involved in transcriptional regulation are transparent without a white background.
Interactions identified in vitro between cyclins and CDKs are indicated by a dashed orange arrow. Table 2 identifies all sources used to construct the figure. The cell cycles at sporogony and hepatic schizogony are omitted due to the lack of information about these stages. None of the cyclins are homologs of canonical cell-cycle cyclins e. Although PK5 is the putative homologue of mammalian CDK1, Plasmodium encodes no cognate cyclins for such an enzyme and the activator for PK5 remains unknown: in vitro , it is unusually promiscuous and can be activated by all three Plasmodium cyclins as well as mammalian p25, cyclin H and RINGO [ 37 , 44 , 45 ].
The partnership with Cyc1 is questionable because recent immunoprecipitation studies failed to identify it [ 46 ].
Mrk1 is not apparently a CDK-activating kinase in Plasmodium and although it can interact in vitro with the replication factor complex PfRFC-5 and PfMCM6 [ 40 ], it actually appears to be crucial for cytokinesis rather than replication, as indeed is Cyc1 [ 46 ], with Mrk1 acting in a complex with Cyc1 and MAT1 [ 46 ]. Crk5 can be activated in vitro by Cyc1 and Cyc4 but its in vivo partner is again unknown; it is involved in, but not essential for, erythrocytic schizogony because its absence results in viable parasites with fewer merozoites per schizont [ 43 ].
PK6 is proposed to be involved in the onset of S phase in erythrocytic stages but in vivo characterisation is lacking, and recombinant PK6 is cyclin-independent in vitro [ 47 ]. The remaining CDKs, Crk-1 and Crk-3, are predicted to have roles in transcriptional regulation and thus in cell growth and proliferation [ 42 , 48 ].
Another - perhaps more interesting - group of regulators are specific to the unusual cell cycle modes of apicomplexans and there is considerable interest in the plant-like calcium-dependent protein kinases CDPKs as possible parasite-specific drug targets, with CDPK4 playing multiple roles in male gametogenesis [ 28 ] and CDPK7, in erythrocytic schizogony [ 53 ]. Another Plasmodium -specific kinase, PfCRK4, was recently identified as essential for DNA replication in erythrocytic schizogony, although the pathway in which it acts remains to be elucidated [ 12 ].
In addition to the cyclin-CDK regulatory network, there are also defined checkpoints in yeast and mammalian systems that control cell cycle advancement. These serve as quality control for cell growth G1 checkpoint , successful DNA replication or DNA damage S and G2 checkpoints and chromosome attachment to the spindle M checkpoint [ 54 ].
Checkpoints are particularly important for avoiding re-replication and preventing the propagation of incompletely replicated or damaged daughter genomes. The existence of cell cycle checkpoints in Plasmodium remains, in general, uncertain, and genes encoding key checkpoint proteins such as Rb, p53, ATM and ATR have not been identified. There is, however, some evidence of a G1 checkpoint in the related parasite T.
What induces these states in the absence of a conventional G1 checkpoint pathway is unclear. DNA repair machinery is largely conserved in the parasite genome, as described below, and parasites respond to DNA damage by upregulating repair machinery and altering chromosome structure [ 60 ]. However, there is no apparent G2, offering no opportunity for a G2 checkpoint [ 13 , 35 ] and the feasibility of intra-S and M checkpoints is challenged by the striking variation in the speed of genome replication at different life-cycle stages, particularly the unprecedented rate in male gametocytes [ 61 ], sharply contrasting with a more conventional rate during erythrocytic schizogony [ 13 , 31 ].
Checkpoint regulation may be temporally possible during erythrocytic schizogony - and perhaps also oocyst sporogony and hepatic schizogony - but not gametogenesis. Spatially, schizogony also poses challenges to checkpoint control. Although chromosomes do appear to align with the hemispindle, which is anchored to a CP, they remain uncondensed: it is thought that the centromeres remain constantly attached to CPs and that this may help to separate the uncondensed chromosomes accurately [ 3 ].
Finally, the syncytial nature of Plasmodium replication raises questions about diffusible checkpoint factors and the how the replication of individual genomes could be stalled within a shared cytoplasm [ 2 ]. Variations in cell cycle speed also raise questions about replicative fidelity and tolerance of karyotypic variation. Under drug pressure, P. This permits fine-tuning of amplicon numbers, relevant to drug pressure, while avoiding genome damage and any deleterious mutations in off-target loci [ 63 ].
Two recent studies have suggested that the mitotic mutation rate does not vary between P. In this regard, P. The extremely fast replication of male gametes raises a particular conundrum in terms of checkpoints: does this phase require especially stringent regulation, or conversely, more relaxed control to favour speed over fidelity? The observation that some male gametes are produced with apparently partial or absent nuclei unpublished observations and the fact that male-expressed genes display fast rates of evolution [ 71 ] may suggest the latter.
Proteins involved in DNA replication and mitosis are simultaneously phosphorylated within the first 20 seconds of gametocyte activation, contrary to the traditional view of sequential progression through the cell cycle, and this may facilitate the rapidity of gametogenesis [ 32 ]. Indeed, the relatively limited repertoire of cell cycle kinases in Plasmodium may also imply that some have dual functions: CDPK4 has been implicated in assembly of the pre-replicative complex, mitotic spindle formation, cytokinesis and axoneme motility [ 28 , 32 , 72 ].
Regardless of cell cycle speed, the parasite is clearly able to promote genomic diversity during mitosis, as well as more conventionally at meiosis. It seems unlikely that the intricacy and precision of the Plasmodium cell cycles would proceed unchecked, but evidence is currently lacking for clearly defined checkpoints and there may be great flexibility in which checkpoints are enforced during different types of replication. DNA damage can originate from a range of sources, the most common in Plasmodium being reactive oxygen species generated by metabolism, free radicals, which are often produced after uptake of antimalarial drugs such as chloroquine or artemisinin, and errors made during DNA replication.
Damage may affect individual bases or may lead to the generation of potentially deadly double strand breaks DSBs. The mutational spectrum observed in P. This can promote the formation of pseudopolyploid loci, as described above, but the core genome nevertheless remains intact, due to the presence of an effective DNA repair system including most - although notably not all - of the pathways commonly found in model eukaryotes.
Orthologs of the majority of genes involved in the NER pathway have been identified bioinformatically, with the exception of p62 and XPC [ 73 ]. Similarly, the majority of the MMR pathway is present but there are notable differences from other eukaryotes, with RecJ exonucleases appearing to be absent while a UvrD helicase homolog, found in E. The majority of eukaryotes rely upon two major pathways for the repair of double-strand breaks, homologous recombination HR and non-homologous end joining NHEJ.
The Plasmodium genome encodes a functional HR pathway but the core genes of the NHEJ pathway appear to be absent across the genus [ 21 , 77 ], supported by the inability to detect NHEJ products in vitro after the experimental generation of DSBs [ 78 , 79 ]. HR, indeed, appears to be essential for the completion of the parasite life-cycle because the knockout of a zinc finger protein, Pb Zfp, in P.
During all haploid growth phases the parasite must therefore rely upon alternative end joining pathways such as microhomology-mediated end joining MMEJ to repair DSBs within the core genome, because no repair template exists to allow HR [ 84 ]. Bioinformatic comparisons with the S. Notably, this restriction does not apply to multigene families, such as the var family of key virulence genes in P.
This leads to important diversification of these gene families during mitotic growth [ 68 ] as well as during meiosis , generating new antigenic variants that can facilitate immune evasion during chronic human infections. Var gene recombination does not require substantial stretches of high sequence homology and the genes do not necessarily recombine with their closest homologues [ 68 ]; the physical clustering of var genes at the nuclear periphery may favour sequence pairing even in the absence of extensive homology.
Historically, DNA replication has been an excellent drug target in malaria parasites, as demonstrated by Fansidar: an anti-folate drug combination which proved vital after the emergence of chloroquine resistance in the late s [ 88 ]. Fansidar is a synergistic combination of two drugs that block the pathway to production of reduced folate cofactors essential for nucleotide production and DNA synthesis , but resistance to the combination arose fairly rapidly [ 89 , 90 ].
However, directly targeting the regulatory machinery of the parasite, such as cell-cycle checkpoint control, or eliciting DNA damage as a route to parasite killing, may provide a greater hurdle to resistance development. Indeed, DNA damage, together with protein damage, is thought to be a mode of action for the frontline antimalarial drug artemisinin, mediated through free radicals [ 91 ]. For Plasmodium , understanding the cell cycle arrest phenotype takes on new urgency because it is considered a key mechanism of artemisinin resistance [ 94 ].
This resistance is not yet fully understood in molecular terms, but it correlates with mutations in the Kelch protein, which in turn correlate with elevated levels of the phosphoinositidekinase enzyme PfPI3K [ 95 ].
PfPI3K, a lipid kinase, is distantly related to protein kinases that are key checkpoint proteins in most eukaryotes but are missing in Plasmodium - an intriguing similarity that is currently under investigation in our laboratory.
The ability of resistant parasites to survive in a dormant state and recrudesce weeks later may be exacerbated by the recent finding that erythrocytic schizogony is remarkably flexible in resistant parasites. Resistant clones from Southeast Asia have a prolonged ring stage and considerably shortened trophozoite stage, presumably reducing drug exposure to the more vulnerable trophozoite [ 57 ].
This phenotype was stable without artemisinin drug pressure, which is particularly worrying because it could reduce the available window for antimalarials that target the trophozoite stage and that may be used as partner drugs in artemisinin combination therapies, or as alternative drugs if such therapies fail. This is in addition to mutations in the Kelch propeller domain [ 96 ], which serves as a molecular marker for resistance. In bacterial systems, mild mutators acquire mutations at a lower rate than hypermutator lines but remain able to purge deleterious mutations efficiently from the genome and thus they can outcompete hypermutators in the long term [ 97 , 98 , 99 , ].
A fine balance is clearly required between the need for accuracy and adaptability, and this balance has probably shifted in P. By analogy with cancer cells - which frequently have deficient checkpoint and DNA repair pathways - artemisinin resistant parasites may be on a knife edge between efficient growth and the potential disaster of an under-regulated cell cycle, so it may be possible to target them with drugs that exacerbate their defects. What is DNA replication? See all questions in DNA Replication.
Impact of this question views around the world. You can reuse this answer Creative Commons License. Cell Division and Cancer. Cytokinesis Mechanisms in Yeast. Recovering a Stalled Replication Fork. Aging and Cell Division. Germ Cells and Epigenetics. Citation: Das-Bradoo, S. Nature Education 3 9 During DNA replication, the unwinding of strands leaves a single strand vulnerable.
How does the cell protect these strands from damage? Aa Aa Aa. Figure 1: The major replication events in a prokaryotic cell. A Nucleoside triphosphates serve as a substrate for DNA polymerase, according to the mechanism shown on the top strand. The Leading and Lagging Strands. These proteins are illustrated schematically in panel a of the figure below, but in reality, the fork is folded in three dimensions, producing a structure resembling that of the diagram in the inset b.
Triggering a Checkpoint. Other Roles for ATR. Stalled Forks. Nucleases can cleave stalled forks, causing double-strand breaks DSBs to form and activate ataxia-telangiectasia mutated ATM. References and Recommended Reading Alberts, B. Article History Close. Share Cancel. Revoke Cancel. Keywords Keywords for this Article. Save Cancel. Flag Inappropriate The Content is: Objectionable. Flag Content Cancel.
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