Abstract:The mechanics of DNA replication and cell cycling are well-characterized in model organisms, but less is known about these basic aspects of cell biology in early-diverging Apicomplexan parasites, which do not divide by canonical binary fission but undergo unconventional cycles. Schizogony in the malaria parasite, Plasmodium, generates ~16–24 new nuclei via independent, asynchronous rounds of genome replication prior to cytokinesis and little is known about the control of DNA replication that facilitates this. … Show more
“…DNA ‘combing’ of labelled DNA fibres was recently used to investigate the actual distribution of active replication forks during erythrocytic schizogony (Fig. 3c ) and this revealed a mean distance of 65 kb between individual origins, with replication forks moving at a mean rate of 1.19 kb/min [ 31 ]. 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.…”
Section: Mechanics Of Dna Replication At the Molecular Levelmentioning
confidence: 99%
“…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. In a schizont, which is replicating not 2-fold but ~ 16-fold, the availability of nucleotides, the physical space available or the increasing compaction of chromatin may all limit the speed of replication as S phase advances [ 31 ]. Fig.…”
Section: Mechanics Of Dna Replication At the Molecular Levelmentioning
confidence: 99%
“…This may include origins that would never be activated, and may miss those where the protein complex has dissociated from the chromosome. c Synthetic nucleoside labelling and DNA combing techniques allow the labelling and fluorescent immunodetection of de novo DNA synthesis [ 31 ]. 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.…”
Section: Mechanics Of Dna Replication At the Molecular Levelmentioning
confidence: 99%
“…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.…”
Section: Cell Cycle Checkpoints In
Plasmodiummentioning
It is over 100 years since the life-cycle of the malaria parasite Plasmodium was discovered, yet its intricacies remain incompletely understood - a knowledge gap that may prove crucial for our efforts to control the disease. Phenotypic screens have partially filled the void in the antimalarial drug market, but as compound libraries eventually become exhausted, new medicines will only come from directed drug development based on a better understanding of fundamental parasite biology. This review focusses on the unusual cell cycles of Plasmodium, which may present a rich source of novel drug targets as well as a topic of fundamental biological interest. Plasmodium does not grow by conventional binary fission, but rather by several syncytial modes of replication including schizogony and sporogony. Here, we collate what is known about the various cell cycle events and their regulators throughout the Plasmodium life-cycle, highlighting the differences between Plasmodium, model organisms and other apicomplexan parasites and identifying areas where further study is required. The possibility of DNA replication and the cell cycle as a drug target is also explored. Finally the use of existing tools, emerging technologies, their limitations and future directions to elucidate the peculiarities of the Plasmodium cell cycle are discussed.
“…DNA ‘combing’ of labelled DNA fibres was recently used to investigate the actual distribution of active replication forks during erythrocytic schizogony (Fig. 3c ) and this revealed a mean distance of 65 kb between individual origins, with replication forks moving at a mean rate of 1.19 kb/min [ 31 ]. 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.…”
Section: Mechanics Of Dna Replication At the Molecular Levelmentioning
confidence: 99%
“…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. In a schizont, which is replicating not 2-fold but ~ 16-fold, the availability of nucleotides, the physical space available or the increasing compaction of chromatin may all limit the speed of replication as S phase advances [ 31 ]. Fig.…”
Section: Mechanics Of Dna Replication At the Molecular Levelmentioning
confidence: 99%
“…This may include origins that would never be activated, and may miss those where the protein complex has dissociated from the chromosome. c Synthetic nucleoside labelling and DNA combing techniques allow the labelling and fluorescent immunodetection of de novo DNA synthesis [ 31 ]. 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.…”
Section: Mechanics Of Dna Replication At the Molecular Levelmentioning
confidence: 99%
“…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.…”
Section: Cell Cycle Checkpoints In
Plasmodiummentioning
It is over 100 years since the life-cycle of the malaria parasite Plasmodium was discovered, yet its intricacies remain incompletely understood - a knowledge gap that may prove crucial for our efforts to control the disease. Phenotypic screens have partially filled the void in the antimalarial drug market, but as compound libraries eventually become exhausted, new medicines will only come from directed drug development based on a better understanding of fundamental parasite biology. This review focusses on the unusual cell cycles of Plasmodium, which may present a rich source of novel drug targets as well as a topic of fundamental biological interest. Plasmodium does not grow by conventional binary fission, but rather by several syncytial modes of replication including schizogony and sporogony. Here, we collate what is known about the various cell cycle events and their regulators throughout the Plasmodium life-cycle, highlighting the differences between Plasmodium, model organisms and other apicomplexan parasites and identifying areas where further study is required. The possibility of DNA replication and the cell cycle as a drug target is also explored. Finally the use of existing tools, emerging technologies, their limitations and future directions to elucidate the peculiarities of the Plasmodium cell cycle are discussed.
“…Nonetheless, since we normalize our quantitation of fork-stalling to an untreated sample, our results are internally controlled. Furthermore, double-labeled combing studies done in mammalian cells, Plasmodium falciparum , and Trypansoma brucei show similar rate of fork stalling in the untreated sample (Merav Socolovsky, personal communication) [ 91 , 92 ].…”
In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.
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