Saccharomyces cerevisiae
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PROVIDER: PRJNA141229 | ENA |
REPOSITORIES: ENA
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Project description:Initiation of eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) initially drives origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Corresponding in vivo studies found that DDK was required for Cdc45 binding at early origins during G1. Upon activation of S-phase cyclin-dependent kinases (S-CDK), a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Investigation of DNA polymerase recruitment showed that Mcm10 and DNA unwinding both were critical for recruitment of the lagging but not leading strand DNA polymerases. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which the leading strand DNA polymerase is recruited prior to DNA unwinding and initial RNA primer synthesis. We performed chromatin immunoprecipitation (ChIP) against Cdc45 in CDC7 and cdc7-4 cells arrested in G1 phase to assess the requirement of the Dbf4-dependent kinase on the recruitment of Cdc45 to origin DNA during G1.
2011-07-21 | E-GEOD-29646 | biostudies-arrayexpress
Project description:Initiation of eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) initially drives origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Corresponding in vivo studies found that DDK was required for Cdc45 binding at early origins during G1. Upon activation of S-phase cyclin-dependent kinases (S-CDK), a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Investigation of DNA polymerase recruitment showed that Mcm10 and DNA unwinding both were critical for recruitment of the lagging but not leading strand DNA polymerases. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which the leading strand DNA polymerase is recruited prior to DNA unwinding and initial RNA primer synthesis.
2011-07-22 | GSE29646 | GEO
Project description:Progress through the division cycle of present day eukaryotic cells is controlled by a complex network consisting of (i) cyclin-dependent kinases (CDKs) and their associated cyclins, (ii) kinases and phosphatases that regulate CDK activity, and (iii) stoichiometric inhibitors that sequester cyclin-CDK dimers. Presumably regulation of cell division in the earliest ancestors of eukaryotes was a considerably simpler affair. Nasmyth (1995) recently proposed a mechanism for control of a putative, primordial, eukaryotic cell cycle, based on antagonistic interactions between a cyclin-CDK and the anaphase promoting complex (APC) that labels the cyclin subunit for proteolysis. We recast this idea in mathematical form and show that the model exhibits hysteretic behaviour between alternative steady states: a Gl-like state (APC on, CDK activity low, DNA unreplicated and replication complexes assembled) and an S/M-like state (APC off, CDK activity high, DNA replicated and replication complexes disassembled). In our model, the transition from G1 to S/M ('Start') is driven by cell growth, and the reverse transition ('Finish') is driven by completion of DNA synthesis and proper alignment of chromosomes on the metaphase plate. This simple and effective mechanism for coupling growth and division and for accurately copying and partitioning a genome consisting of numerous chromosomes, each with multiple origins of replication, could represent the core of the eukaryotic cell cycle. Furthermore, we show how other controls could be added to this core and speculate on the reasons why stoichiometric inhibitors and CDK inhibitory phosphorylation might have been appended to the primitive alternation between cyclin accumulation and degradation.
2020-05-04 | MODEL2005040001 | BioModels
Project description:Cell-cycle transitions are generally triggered by variations in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have evolved unique cell-cycles with a repertoire of ancestral CDKs and cyclins whose functions and interdependency remain elusive. Here, we show that the divergent Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical cell-cycle regulator of gametogony required for transmission to the mosquito. It phosphorylates canonical CDK motifs on components of the pre-replicative complex and is essential for DNA replication. We also provide evidence for indirect regulation of the concomitant progression through M-phase. Over a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2) with no evidence of SOC2 cycling through transcription, translation nor degradation. Our results present evidence that during Plasmodium gametogony, a unique and divergent cyclin/CDK pair evolved to fulfil the functional space of multiple eukaryotic cell-cycle kinases controlling S-phase entry and progression through M-phase.
2020-06-17 | GSE144743 | GEO
Project description:Cell-cycle transitions are generally triggered by variations in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria parasites express ancestral CDKs and cyclins, whose functions and interdependency remain elusive. Here, we show that the unique Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical cell-cycle regulator of gametogony required for transmission to the mosquito. It is essential for DNA replication and phosphorylates canonical S/TPxK CDK motifs on components of the pre-replicative complex otherwise regulated by distinct kinases in other eukaryotes. Over a replicative cycle, CRK5 stably interacts with a single Haemosporidia-specific cyclin (SOC2) with no evidence of SOC2 degradation. Regulation of CRK5 activity relies instead on dynamic phosphorylation of a C-terminus extension that mediates its interaction with SOC2. Our results present evidence that during the atypical cell cycles of Plasmodium gametogony, a unique and divergent cyclin/CDK pair fulfils the functional space of multiple eukaryotic cell-cycle kinases to initiate DNA replication.
2020-02-18 | PXD017283 | Pride
Project description:Genome stability requires complete DNA duplication exactly once before division. In eukaryotes, cyclin-dependent kinase (CDK) plays a dual role in this regulation by inhibiting helicase loading factors and activating origin firing. CDK drives initiation by phosphorylation of two substrates, Sld2 and Sld3, forming the transient and limiting intermediate called the pre-initiation complex (pre-IC). The importance and mechanism of dissociation of the pre-IC from origins is not understood. Here we show in the budding yeast Saccharomyces cerevisiae that CDK phosphorylations of Sld3 and Sld2 are specifically and rapidly turned-over during interphase by the PP2A and PP4 phosphatases. Inhibition of dephosphorylation of Sld3/Sld2 causes dramatic defects in replication initiation genome-wide, retention of the pre-IC at origins and cell death. These studies not only provide a mechanism to ensure that Sld3/Sld2 are dephosphorylated before helicase loading factors but also establish a novel positive role for phosphatases in eukaryotic origin firing.
2021-11-01 | GSE186490 | GEO
Project description:Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. We also provide evidence for indirect regulation of the concomitant M-phase progression. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this unique cyclin/CDK pair fills the functional space of multiple eukaryotic cell-cycle kinases controlling DNA replication and M-phase progression.
2020-06-17 | PXD017622 | Pride
Project description:We propose an integrated computational model for the network of cyclin-dependent kinases (Cdks) that controls the dynamics of the mammalian cell cycle. The model contains four Cdk modules regulated by reversible phosphorylation, Cdk inhibitors, and protein synthesis or degradation. Growth factors (GFs) trigger the transition from a quiescent, stable steady state to self-sustained oscillations in the Cdk network. These oscillations correspond to the repetitive, transient activation of cyclin D/Cdk4-6 in G(1), cyclin E/Cdk2 at the G(1)/S transition, cyclin A/Cdk2 in S and at the S/G(2) transition, and cyclin B/Cdk1 at the G(2)/M transition. The model accounts for the following major properties of the mammalian cell cycle: (i) repetitive cell cycling in the presence of suprathreshold amounts of GF; (ii) control of cell-cycle progression by the balance between antagonistic effects of the tumor suppressor retinoblastoma protein (pRB) and the transcription factor E2F; and (iii) existence of a restriction point in G(1), beyond which completion of the cell cycle becomes independent of GF. The model also accounts for endoreplication. Incorporating the DNA replication checkpoint mediated by kinases ATR and Chk1 slows down the dynamics of the cell cycle without altering its oscillatory nature and leads to better separation of the S and M phases. The model for the mammalian cell cycle shows how the regulatory structure of the Cdk network results in its temporal self-organization, leading to the repetitive, sequential activation of the four Cdk modules that brings about the orderly progression along cell-cycle phases.
2024-09-02 | BIOMD0000000730 | BioModels
Project description:Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal DNA, triggering a series of molecular events that culminate in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome requires multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern with relevance to differentiation, genome stability and evolution. It is unclear whether ORC-origin interactions are relevant to the regulation of origin activation time. Here we applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. The two classes of origins were distinct in terms of local nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Importantly, origins that fired in late S-phase, but not those that fired in early S-phase, showed a significant dependence on the ORC-origin DNA interaction for ORC binding in vivo. Therefore we demonstrate for the first time a connection between ORC-origin binding mechanisms and the regulation of origin activation time at a genome-wide level. Analysis of ORC genomic DNA binding across three ORC concentrations.
2013-09-30 | E-GEOD-48440 | biostudies-arrayexpress
Project description:Cell-cycle transitions are generally triggered by variations in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have evolved unique cell-cycles with a repertoire of ancestral CDKs and cyclins whose functions and interdependency remain elusive. Here, we show that the divergent Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical cell-cycle regulator of gametogony required for transmission to the mosquito. It phosphorylates canonical CDK motifs on components of the pre-replicative complex and is essential for DNA replication. We also provide evidence for indirect regulation of the concomitant progression through M-phase. Over a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2) with no evidence of SOC2 cycling through transcription, translation nor degradation. Our results present evidence that during Plasmodium gametogony, a unique and divergent cyclin/CDK pair evolved to fulfil the functional space of multiple eukaryotic cell-cycle kinases controlling S-phase entry and progression through M-phase.
2020-02-25 | PXD017308 | Pride