<jats:p>In<jats:italic>Escherichia coli</jats:italic>, replisome and replication fork assembly is initiated by DnaB helicase loading at the chromosomal origin<jats:italic>oriC</jats:italic>via its interactions with the DnaA initiator and the DnaC helicase loader. Upon replication fork arrest, the replisome including DnaB dissociates from the stalled fork. Replication fork progression is rescued by primosomal protein PriA- or PriC-dependent pathway in which PriA and PriC promotes reloading of DnaB in different mechanisms. However, the mechanism responsible for rescue of blocked replication initiation at<jats:italic>oriC</jats:italic>remains unclear. Here, we found that PriC rescued blocked replication initiation in cells expressing an initiation-specific DnaC mutant, in mutant cells defective in DnaA-DnaB interactions, and in cells containing truncated<jats:italic>oriC</jats:italic>sequence variants. PriC rescued DnaB loading at<jats:italic>oriC</jats:italic>even in the absence of Rep helicase, a stimulator of the PriC-dependent replication fork restart pathway. These and results of<jats:italic>in vitro</jats:italic>reconstituted assays concordantly suggest that this initiation-specific rescue mechanism provides a bypass of the DnaA-DnaB interaction for DnaB loading by PriC-promoted loading of DnaB to the unwound<jats:italic>oriC</jats:italic>region. These findings expand understanding of mechanisms sustaining the robustness of replication initiation and specific roles for PriC in the genome maintenance.</jats:p>

DOI： 10.1101/2024.10.06.616894

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Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host’s advantage in acquiring a viral helicase loader gene.

DOI： 10.1073/pnas.2317954121

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media  

Timely initiation of chromosomal DNA replication in Escherichia coli is achieved by cell cycle-coordinated regulation of the replication origin, oriC, and the replication initiator, ATP-DnaA. Cellular levels of ATP-DnaA increase and peak at the time for initiation at oriC, after which hydrolysis of DnaA-bound ATP causes those to fall, yielding initiation-inactive ADP-DnaA. This hydrolysis is facilitated by the chromosomal locus datA located downstream of the tRNA-Gly (glyV-X-Y) operon, which possesses a cluster of DnaA-binding sequences and a single binding site (IBS) for the DNA bending protein IHF (integration host factor). While IHF binding activates the datA function and is regulated to occur specifically at post-initiation time, the underlying regulatory mechanisms remain obscure. Here, we demonstrate that datA-IHF binding at pre-initiation time is down-regulated depending on the read-through transcription of datA IBS initiated at the glyV-X-Y promoter. During the cell cycle, the level of read-through transcription, but not promoter activity, fluctuated in a manner inversely related to datA-IHF binding. Transcription from the glyV-X-Y promoter was predominantly interrupted at datA IBS by IHF binding. The terminator/attenuator sequence of the glyV-X-Y operon, as well as DnaA binding within datA overall, contributed to attenuation of transcription upstream of datA IBS, preserving the timely fluctuation of read-through transcription. These findings provide a mechanistic insight of tRNA transcription-dependent datA-IHF regulation, in which an unidentified factor is additionally required for the timely datA-IHF dissociation, and support the significance of datA for controlling the cell cycle progression as a connecting hub of tRNA production and replication initiation.

DOI： 10.3389/fmicb.2024.1360108

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Language：English   Publisher：American Society for Biochemistry and Molecular Biology  

Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA–DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded (ss) DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and ssDUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA–DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC was also responsible for unwinding. These findings indicate that a ssDUE recruitment mechanism was responsible for unwinding and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

This review summarizes current knowledge about the mechanisms of timely binding and dissociation of two nucleoid proteins, IHF and Fis, which play fundamental roles in the initiation of chromosomal DNA replication in Escherichia coli. Replication is initiated from a unique replication origin called oriC and is tightly regulated so that it occurs only once per cell cycle. The timing of replication initiation at oriC is rigidly controlled by the timely binding of the initiator protein DnaA and IHF to oriC. The first part of this review presents up-to-date knowledge about the timely stabilization of oriC-IHF binding at oriC during replication initiation. Recent advances in our understanding of the genome-wide profile of cell cycle-coordinated IHF binding have revealed the oriC-specific stabilization of IHF binding by ATP-DnaA oligomers at oriC and by an initiation-specific IHF binding consensus sequence at oriC. The second part of this review summarizes the mechanism of the timely regulation of DnaA activity via the chromosomal loci DARS2 (DnaA-reactivating sequence 2) and datA. The timing of replication initiation at oriC is controlled predominantly by the phosphorylated form of the adenosine nucleotide bound to DnaA, i.e., ATP-DnaA, but not ADP-ADP, is competent for initiation. Before initiation, DARS2 increases the level of ATP-DnaA by stimulating the exchange of ADP for ATP on DnaA. This DARS2 function is activated by the site-specific and timely binding of both IHF and Fis within DARS2. After initiation, another chromosomal locus, datA, which inactivates ATP-DnaA by stimulating ATP hydrolysis, is activated by the timely binding of IHF. A recent study has shown that ATP-DnaA oligomers formed at DARS2-Fis binding sites competitively dissociate Fis via negative feedback, whereas IHF regulation at DARS2 and datA still remains to be investigated. This review summarizes the current knowledge about the specific role of IHF and Fis in the regulation of replication initiation and proposes a mechanism for the regulation of timely IHF binding and dissociation at DARS2 and datA.

DOI： 10.3390/ijms241411572

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nucleic Acids Research  

The Escherichia coli replication origin oriC contains the initiator ATP-DnaA-Oligomerization Region (DOR) and its flanking duplex unwinding element (DUE). In the Left-DOR subregion, ATP-DnaA forms a pentamer by binding to R1, R5M and three other DnaA boxes. The DNA-bending protein IHF binds sequence-specifically to the interspace between R1 and R5M boxes, promoting DUE unwinding, which is sustained predominantly by binding of R1/R5M-bound DnaAs to the single-stranded DUE (ssDUE). The present study describes DUE unwinding mechanisms promoted by DnaA and IHF-structural homolog HU, a ubiquitous protein in eubacterial species that binds DNA sequence-non-specifically, preferring bent DNA. Similar to IHF, HU promoted DUE unwinding dependent on ssDUE binding of R1/R5M-bound DnaAs. Unlike IHF, HU strictly required R1/R5M-bound DnaAs and interactions between the two DnaAs. Notably, HU site-specifically bound the R1-R5M interspace in a manner stimulated by ATP-DnaA and ssDUE. These findings suggest a model that interactions between the two DnaAs trigger DNA bending within the R1/R5M-interspace and initial DUE unwinding, which promotes site-specific HU binding that stabilizes the overall complex and DUE unwinding. Moreover, HU site-specifically bound the replication origin of the ancestral bacterium Thermotoga maritima depending on the cognate ATP-DnaA. The ssDUE recruitment mechanism could be evolutionarily conserved in eubacteria.

DOI： 10.1093/nar/gkad389

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Unwinding of the replication origin and loading of DNA helicases underlie the initiation of chromosomal replication. In Escherichia coli, the minimal origin oriC contains a duplex unwinding element (DUE) region and three (Left, Middle, and Right) regions that bind the initiator protein DnaA. The Left/Right regions bear a set of DnaA-binding sequences, constituting the Left/Right-DnaA subcomplexes, while the Middle region has a single DnaA binding site, which stimulates formation of the Left/Right-DnaA subcomplexes. In addition, a DUE-flanking AT-cluster element (TATTAAAAAGAA) is located just outside of the minimal oriC region. The Left-DnaA subcomplex promotes unwinding of the flanking DUE exposing TT[A/G]T(T) sequences that then bind to the Left-DnaA subcomplex, stabilizing the unwound state required for DnaB helicase loading. However, the role of the Right-DnaA-subcomplex is largely unclear. Here, we show that DUE unwinding by both the Left/Right-DnaA complexes, but not the Left-DnaA subcomplex only, was stimulated by a DUE-terminal subregion flanking the AT-cluster. Consistently, we found the Right-DnaA subcomplex bound single-stranded DUE and AT-cluster regions. In addition, the Left/Right-DnaA subcomplexes bound DnaB helicase independently. For only the Left-DnaA subcomplex, we show the AT-cluster was crucial for DnaB loading. The role of unwound DNA binding of the Right-DnaA subcomplex was further supported by in vivo data. Taken together, we propose a model in which the Right-DnaA subcomplex dynamically interacts with the unwound DUE, assisting in DUE unwinding and efficient loading of DnaB helicases, while in the absence of the Right-DnaA subcomplex, the AT-cluster assists in those processes, supporting robustness of replication initiation.

DOI： 10.1016/j.jbc.2022.102051

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DOI： 10.1093/nar/gkab1171

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<title>Abstract</title>Nucleotide-based signaling molecules (NSMs) are widespread in bacteria and eukaryotes, where they control important physiological and behavioral processes. In bacteria, NSM-based regulatory networks are highly complex, entailing large numbers of enzymes involved in the synthesis and degradation of active signaling molecules. How the converging input from multiple enzymes is transformed into robust and unambiguous cellular responses has remained unclear. Here we show that <italic>Escherichia coli</italic> converts dynamic changes of c-di-GMP into discrete binary signaling states, thereby generating heterogeneous populations with either high or low c-di-GMP. This is mediated by an ultrasensitive switch protein, PdeL, which senses the prevailing cellular concentration of the signaling molecule and couples this information to c-di-GMP degradation and transcription feedback boosting its own expression. We demonstrate that PdeL acts as a digital filter that facilitates precise developmental transitions, confers cellular memory, and generates functional heterogeneity in bacterial populations to evade phage predation. Based on our findings, we propose that bacteria apply ultrasensitive regulatory switches to convert dynamic changes of NSMs into binary signaling modes to allow robust decision-making and bet-hedging for improved overall population fitness.

DOI： 10.1101/2021.10.01.462762

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The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via interactions with the DnaC loader and the DnaA complex. The DnaB-DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB-DnaC complex is then directed to unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe46 in DnaA domain I binds to DnaB. Here, we searched for the DnaA domain I-binding site in DnaB. The DnaB L160A variant was impaired in binding to DnaA complex on oriC but retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A onto oriC was consistently and moderately inhibited. In a helicase assay with partly single-stranded DNA bearing a DnaA-binding site, DnaA stimulated DnaB loading, which was strongly inhibited in DnaB L160A even in the presence of DnaC. DnaB L160A was functionally impaired in vivo On the basis of these findings, we propose that DnaB Leu160 interacts with DnaA domain I Phe46 DnaB Leu160 is exposed on the lateral surface of the N-terminal domain, which can explain unobstructed interactions of DnaA domain I-bound DnaB with DnaC, DnaG primase, and DnaA domain III. We propose a probable structure for the DnaA-DnaB-DnaC complex, which could be relevant to the process of DnaB loading onto oriC.

DOI： 10.1074/jbc.RA120.014235

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ATP-DnaA is temporally increased to initiate replication during the cell cycle. Two chromosomal loci, DARS (DnaA-reactivating sequences) 1 and 2, promote ATP-DnaA production by nucleotide exchange of ADP-DnaA for timely initiation. ADP-DnaA complexes are constructed on DARS1 and DARS2, bearing a cluster of three DnaA-binding sequences (DnaA boxes I-III), promoting ADP dissociation. Although DnaA has an AAA+ domain, which ordinarily directs construction of oligomers in a head-to-tail manner, DnaA boxes I and II are oriented oppositely. In this study, we constructed a structural model of a head-to-head dimer of DnaA AAA+ domains, and analyzed residues residing on the interface of the model dimer. Gln208 was specifically required for DARS-dependent ADP dissociation in vitro, and in vivo analysis yielded consistent results. Additionally, ADP release from DnaA protomers bound to DnaA boxes I and II was dependent on Gln208 of the DnaA protomers, and DnaA box III-bound DnaA did not release ADP nor require Gln208 for ADP dissociation by DARS-DnaA complexes. Based on these and other findings, we propose a model for DARS-DnaA complex dynamics during ADP dissociation, and provide novel insight into the regulatory mechanisms of DnaA and the interaction modes of AAA+ domains.

DOI： 10.1093/nar/gkz795

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Chromosome replication is a fundamental process in all domains of life. To accurately transmit genetic material to offspring, the initiation of chromosome replication is tightly regulated to ensure that it occurs only once in each cell division cycle. In the model bacterium Caulobacter crescentus, the CtrA response regulator inhibits the origin of replication at the pre-replication stage. Inactivation of CtrA permits the universal DnaA initiator to form an initiation complex at the origin, leading to replication initiation. Subsequently, the initiation complex is inac-tivated to prevent extra initiation. Whereas DNA replication occurs periodically in exponentially growing cells, replication initiation is blocked under various stress conditions to halt cell cycle progression until the normal condition is restored or the cells adapt to the stress. Thus, regulating the initiation complex plays an important role in not only driving cell cycle progression, but also maintaining cell integrity under stress. Multiple regulatory signaling pathways controlling CtrA and DnaA have been identified and recent studies have advanced our knowledge of the underlying mechanistic and molecular processes. This review focuses on how bacterial cells control replication initiation, highlighting the latest findings that have emerged from studies in C. crescentus.

DOI： 10.1266/ggs.19-00011

Repository Public URL： http://hdl.handle.net/2324/6617899

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The Escherichia coli CrfC protein is an important regulator of nucleoid positioning and equipartition. Previously we revealed that CrfC homo-oligomers bind the clamp, a DNA-binding subunit of the DNA polymerase III holoenzyme, promoting colocalization of the sister replication forks, which ensures the nucleoid equipartition. In addition, CrfC localizes at the cell pole-proximal loci via an unknown mechanism. Here, we demonstrate that CrfC localizes to the distinct subnucleoid structures termed nucleoid poles (the cell pole-proximal nucleoid-edges) even in elongated cells as well as in wild-type cells. Systematic analysis of the nucleoid-associated proteins (NAPs) and related proteins revealed that HU, the most abundant NAP, and SlmA, the nucleoid occlusion factor regulating the localization of cell division apparatus, promote the specific localization of CrfC foci. When the replication initiator DnaA was inactivated, SlmA and HU were required for formation of CrfC foci. In contrast, when the replication initiation was inhibited with a specific mutant of the helicase-loader DnaC, CrfC foci were sustained independently of SlmA and HU. H-NS, which forms clusters on AT-rich DNA regions, promotes formation of CrfC foci as well as transcriptional regulation of crfC. In addition, MukB, the chromosomal structure mainetanice protein, and SeqA, a hemimethylated nascent DNA region-binding protein, moderately stimulated formation of CrfC foci. However, IHF, a structural homolog of HU, MatP, the replication terminus-binding protein, Dps, a stress-response factor, and FtsZ, an SlmA-interacting factor in cell division apparatus, little or only slightly affected CrfC foci formation and localization. Taken together, these findings suggest a novel and unique mechanism that CrfC localizes to the nucleoid poles in two steps, assembly and recruitment, dependent upon HU, MukB, SeqA, and SlmA, which is stimulated directly or indirectly by H-NS and DnaA. These factors might concordantly affect specific nucleoid substructures. Also, these nucleoid dynamics might be significant in the role for CrfC in chromosome partition.

DOI： 10.3389/fmicb.2019.00072

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Chromosomal replication initiation requires dynamic mechanisms in higher-order nucleoprotein complexes that are constructed at the origin of replication. In Escherichia coli, DnaA molecules construct functional oligomers at the origin oriC, enabling localized unwinding of oriC and stable binding of DnaB helicases via multiple domain I molecules of oriC-bound DnaA. DnaA-bound DnaB helicases are then loaded onto the unwound region of oriC for construction of a pair of replisomes for bidirectional replication. However, mechanisms of DnaB loading to the unwound oriC remain largely elusive. In this study, we determined that His136 of DnaA domain III has an important role in loading of DnaB helicases onto the unwound oriC. DnaA H136A mutant protein was impaired in replication initiation in vivo, and in DnaB loading to the unwound oriC in vitro, whereas the protein fully sustained activities for oriC unwinding and DnaA domain I-dependent stable binding between DnaA and DnaB. Functional and structural analyses supported the idea that transient weak interactions between DnaB helicase and DnaA His136 within specific protomers of DnaA oligomers direct DnaB to a region in close proximity to single stranded DNA at unwound oriC bound to DnaA domain III of the DnaA oligomer. The aromatic moiety of His136 is basically conserved at corresponding residues of eubacterial DnaA orthologs, implying that the guidance function of DnaB is common to all eubacterial species.

DOI： 10.3389/fmicb.2018.02017

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Histidine kinases are key components of regulatory networks in bacteria. Although many of these enzymes are bifunctional, mediating both phosphorylation and dephosphorylation of downstream targets, the molecular details of this central regulatory switch are unclear. We showed recently that the universal second messenger cyclic di–guanosine monophosphate (c-di-GMP) drives Caulobacter crescentus cell cycle progression by forcing the cell cycle kinase CckA from its default kinase into phosphatase mode. We use a combination of structure determination, modeling, and functional analysis to demonstrate that c-di-GMP reciprocally regulates the two antagonistic CckA activities through noncovalent cross-linking of the catalytic domain with the dimerization histidine phosphotransfer (DHp) domain. We demonstrate that both c-di-GMP and ADP (adenosine diphosphate) promote phosphatase activity and propose that c-di-GMP stabilizes the ADP-bound quaternary structure, which allows the receiver domain to access the dimeric DHp stem for dephosphorylation. In silico analyses predict that c-di-GMP control is widespread among bacterial histidine kinases, arguing that it can replace or modulate canonical transmembrane signaling.

DOI： 10.1126/sciadv.1600823

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Fundamental to all living organisms is the capacity to coordinate cell division and cell differentiation to generate appropriate numbers of specialized cells. Whereas eukaryotes use cyclins and cyclin-dependent kinases to balance division with cell fate decisions, equivalent regulatory systems have not been described in bacteria. Moreover, the mechanisms used by bacteria to tune division in line with developmental programs are poorly understood. Here we show that Caulobacter crescentus, a bacterium with an asymmetric division cycle, uses oscillating levels of the second messenger cyclic diguanylate (c-di-GMP) to drive its cell cycle. We demonstrate that c-di-GMP directly binds to the essential cell cycle kinase CckA to inhibit kinase activity and stimulate phosphatase activity. An upshift of c-di-GMP during the G1-S transition switches CckA from the kinase to the phosphatase mode, thereby allowing replication initiation and cell cycle progression. Finally, we show that during division, c-di-GMP imposes spatial control on CckA to install the replication asymmetry of future daughter cells. These studies reveal c-di-GMP to be a cyclin-like molecule in bacteria that coordinates chromosome replication with cell morphogenesis in Caulobacter. The observation that c-di-GMP-mediated control is conserved in the plant pathogen Agrobacterium tumefaciens suggests a general mechanism through which this global regulator of bacterial virulence and persistence coordinates behaviour and cell proliferation.

DOI： 10.1038/nature14473

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Intracellular levels of the bacterial second messenger cyclic di-GMP (c-di-GMP) are controlled by antagonistic activities of diguanylate cyclases and phosphodiesterases. The phosphodiesterase PdeH was identified as a key regulator of motility in Escherichia coli, while deletions of any of the other 12 genes encoding potential phosphodiesterases did not interfere with motility. To analyze the roles of E. coli phosphodiesterases, we demonstrated that most of these proteins are expressed under laboratory conditions. We next isolated suppressor mutations in six phosphodiesterase genes, which reinstate motility in the absence of PdeH by reducing cellular levels of c-di-GMP. Expression of all mutant alleles also led to a reduction of biofilm formation. Thus, all of these proteins are bona fide phosphodiesterases that are capable of interfering with different c-di-GMP-responsive output systems by affecting the global c-di-GMP pool. This argues that E. coli possesses several phosphodiesterases that are inactive under laboratory conditions because they lack appropriate input signals. Finally, one of these phosphodiesterases, PdeL, was studied in more detail. We demonstrated that this protein acts as a transcription factor to control its own expression. Motile suppressor alleles led to a strong increase of PdeL activity and elevated pdeL transcription, suggesting that enzymatic activity and transcriptional control are coupled. In agreement with this, we showed that overall cellular levels of c-di-GMP control pdeL transcription and that this control depends on PdeL itself. We thus propose that PdeL acts both as an enzyme and as a c-di-GMP sensor to couple transcriptional activity to the c-di-GMP status of the cell.

DOI： 10.1128/JB.00604-15

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Summary: When Caulobacter crescentus enters S-phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co-option as c-di-GMP effector protein. While the C-terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N-terminal receiver domain, functional adaptation has reversed signal transduction in PopA with the GGDEF domain adopting input function and the receiver domain serving as regulatory output. We show that the N-terminal receiver domain of PopA specifically interacts with RcdA, a component required for CtrA degradation. In contrast, the GGDEF domain serves to target PopA to the cell pole in response to c-di-GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S-phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit components of the CtrA degradation pathway to the protease specific old cell pole of C. crescentus.

DOI： 10.1111/mmi.12777

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Eukaryotic morphogenesis is seeded with the establishment and subsequent amplification of polarity cues at key times during the cell cycle, often using (cyclic) nucleotide signals. We discovered that flagellum de- and repolarization in the model prokaryote Caulobacter crescentus is precisely orchestrated through at least three spatiotemporal mechanisms integrated at TipF. We show that TipF is a cell cycle-regulated receptor for the second messenger-bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP)-that perceives and transduces this signal through the degenerate c-di-GMP phosphodiesterase (EAL) domain to nucleate polar flagellum biogenesis. Once c-di-GMP levels rise at the G1 → S transition, TipF is activated, stabilized, and polarized, enabling the recruitment of downstream effectors, including flagellar switch proteins and the PflI positioning factor, at a preselected pole harboring the TipN landmark. These c-di-GMP-dependent events are coordinated with the onset of tipF transcription in early S phase and together enable the correct establishment and robust amplification of TipF-dependent polarization early in the cell cycle. Importantly, these mechanisms also govern the timely removal of TipF at cell division coincident with the drop in c-di-GMP levels, thereby resetting the flagellar polarization state in the next cell cycle after a preprogrammed period during which motility must be suspended.

DOI： 10.1101/gad.222679.113

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In Escherichia coli, bidirectional chromosomal replication is accompanied by the colocalization of sister replication forks. However, the biological significance of this mechanism and the key factors involved are still largely unknown. In this study, we found that a protein, termed CrfC, helps sustain the colocalization of nascent DNA regions of sister replisomes and promote chromosome equipartitioning. CrfC formed homomultimers that bound to multiple molecules of the clamp, a replisome subunit that encircles DNA, and colocalized with nascent DNA regions in a clamp-binding-dependent manner in living cells. CrfC is a dynamin homolog; however, it lacks the typical membrane-binding moiety and instead possesses a clamp-binding motif. Given that clamps remain bound to DNA after Okazaki fragment synthesis, we suggest that CrfC sustains the colocalization of sister replication forks in a unique manner by linking together the clamp-loaded nascent DNA strands, thereby laying the basis for subsequent chromosome equipartitioning

DOI： 10.1016/j.celrep.2013.07.040

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Background: Multiple DnaA molecules form highly ordered complexes on the origin DNA to initiate chromosomal replication. Results: Novel structural motifs of DnaA are specifically required for the formation of the DNA unwinding-specific DnaA subcomplex. Conclusion: Distinct inter-DnaA interactions are required for the unwinding-specific subcomplex. Significance: Differentiation of the unwinding-specific subcomplex and a key mechanism underlying it are revealed.

DOI： 10.1074/jbc.M112.372052

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In complex with ATP, but not ADP, DnaA protein multimers unwind a specific region of duplex DNA within the chromosomal replication origin, oriC, triggering a series of reactions that result in initiation of DNA replication. Following replication initiation, ATP hydrolysis, which is coupled to DNA replication, results in the generation of initiation-incompetent ADP-DnaA. Suppression of overinitiation of replication requires that ADP-DnaA complexes be stably maintained until the next round of replication. Thus, the functional and structural requirements that ensure stable nucleotide binding to DnaA are crucial for proper regulation of replication. Here, we demonstrate that Glu143 of DnaA, located within the AAA+ box II N-linker motif, is a key residue involved in stable nucleotide binding. A Glu143 substitution variant of DnaA (DnaA E143A) bound to ADP on ice with an affinity similar to wild-type DnaA, but the resultant ADP-DnaA E143A complex was more labile at 37. °C than wild-type ADP-DnaA complexes. Consistent with this, conversion of ADP-DnaA E143A to ATP-DnaA E143A was stimulated at 37. °C in the presence of ATP, which also stimulated replication of a minichromosome in an in vitro reconstitution reaction. Expression of DnaA E143A in vivo inhibited cell growth in an oriC-dependent manner, suggesting that DnaA E143A caused over-initiation of replication, consistent with the in vitro results. Glu is a highly conserved residue at the corresponding position of γ-proteobacterial DnaA orthologs. Our finding of the novel role for the DnaA N-linker region may represent a conserved function of this motif among those DnaA orthologs.

DOI： 10.1016/j.jsb.2012.05.001

Language：English   Publishing type：Research paper (scientific journal)  

In Escherichia coli, the replication origin oriC consists of two functional regions: the duplex unwinding element (DUE) and its flanking DnaA-assembly region (DAR). ATP-DnaA molecules multimerize on DAR, unwinding DUE for DnaB helicase loading. However, DUE-unwinding mechanisms and functional structures in DnaA-oriC complexes supporting those remain unclear. Here, using various in vitro reconstituted systems, we identify functionally distinct DnaA sub-complexes formed on DAR and reveal novel mechanisms in DUE unwinding. The DUE-flanking left-half DAR carrying high-affinity DnaA box R1 and the ATP-DnaA-preferential DnaA box R5, τ1-2 and I1-2 sites formed a DnaA sub-complex competent in DUE unwinding and ssDUE binding, thereby supporting basal DnaB loading activity. This sub-complex is further subdivided into two; the DUE-distal DnaA sub-complex formed on the ATP-DnaA-preferential sites binds ssDUE. Notably, the DUE-flanking, DnaA box R1-DnaA sub-complex recruits DUE to the DUE-distal DnaA sub-complex in concert with a DNA-bending nucleoid protein IHF, thereby promoting DUE unwinding and binding of ssDUE. The right-half DAR-DnaA sub-complex stimulated DnaB loading, consistent with in vivo analyses. Similar features are seen in DUE unwinding of the hyperthermophile, Thermotoga maritima, indicating evolutional conservation of those mechanisms.

DOI： 10.1093/nar/gkr832

Language：English   Publishing type：Research paper (scientific journal)  

Chromosomal replication must be limited to once and only once per cell cycle. This is accomplished by multiple regulatory pathways that govern initiator proteins and replication origins. A principal feature of DNA replication is the coupling of the replication reaction to negative-feedback regulation. Some of the factors that are important in this process have been discovered, including the clamp (DNA polymerase III subunit-Β (DnaN)), the datA locus, SeqA, DnaA homologue protein (Hda) and YabA, as well as factors that are involved at other stages of the regulatory mechanism, such as DnaA initiator-associating protein (DiaA), the DnaA-reactivating sequence (DARS) loci and Soj. Here, we describe the regulation of DnaA, one of the central proteins involved in bacterial DNA replication, by these factors in Escherichia coli, Bacillus subtilis and Caulobacter crescentus.

DOI： 10.1038/nrmicro2314

Language：English   Publishing type：Research paper (scientific journal)  

Escherichia coli DnaA is the initiator of chromosomal replication. Multiple ATP-DnaA molecules assemble at the oriC replication origin in a highly regulated manner, and the resultant initiation complexes promote local duplex unwinding within oriC, resulting in open complexes. DnaB helicase is loaded onto the unwound single-stranded region within oriC via interaction with the DnaA multimers. The tertiary structure of the functional domains of DnaA has been determined and several crucial residues in the initiation process, as well as their unique functions, have been identified. These include specific DNA binding, inter-DnaA interaction, specific and regulatory interactions with ATP and with the unwound single-stranded oriC DNA, and functional interaction with DnaB helicase. An overall structure of the initiation complex is also proposed. These are important for deepening our understanding of the molecular mechanisms that underlie DnaA assembly, oriC duplex unwinding, regulation of the initiation reaction, and DnaB helicase loading. In this review, we summarize recent progress on the molecular mechanisms of the functions of DnaA on oriC. In addition, some members of the AAA+ protein family related to the initiation of replication and its regulation (e.g., DnaA) are briefly discussed.

DOI： 10.1016/j.plasmid.2009.06.003

Language：English   Publishing type：Research paper (scientific journal)  

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.

DOI： 10.1074/jbc.M708684200

Language：English   Publishing type：Research paper (scientific journal)  

Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 Å resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP-DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP-DnaA, and stimulated the assembly of inactive ADP-DnaA-oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP-DnaA-specific- oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP-DnaA-oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner.

DOI： 10.1101/gad.1561207

Language：English   Publishing type：Research paper (scientific journal)  

Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.

DOI： 10.1111/j.1365-2958.2006.05450.x

Language：English   Publishing type：Research paper (scientific journal)  

In Escherichia coli, ATP-DnaA, but not ADP-DnaA, forms an initiation complex that undergoes site-specific duplex DNA unwinding, open complex formation. However, it remains unclear how highly the ATP-dependent activation of the initiation factor is conserved in evolution. The hyperthermophile Thermotoga maritima is one of the most ancient eubacteria in evolution. Here, we show that the DnaA homolog (tmaDnaA) of this bacterium forms open complexes with the predicted origin region (tma-oriC) in vitro. Tma DnaA has a strong and specific affinity for ATP/ADP as well as for 12-mer repeating sequences within the tma-oriC. Unlike ADPtma DnaA, ATPtma DnaA ishighly cooperative in DNA binding and forms open complexes in a manner that depends on temperature and the superhelical tension of the tma-oriC-bearing plasmid. The minimal tma-oriC required for unwinding is a 149-bp region containing five repeats of the 12-mer sequence and two AT-rich 9-mer repeats. Tma DnaA-binding to the 12-mer motif provokes DNA bending. The 9-mer region is the duplex-unwinding site. The tma DnaA-binding and unwinding motifs of tma-oriC share sequence homology with corresponding archaeal and eukaryotic sequences. These findings suggest that the ATP-dependent molecular switch of the initiator and the mechanisms in the replication initiation complex are highly conserved in eubacterial evolution.

DOI： 10.1111/j.1365-2443.2006.00950.x

Language：English   Publishing type：Research paper (scientific journal)  

Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle. In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature-sensitive dnaA508 and dnaA167 mutants at 40 °C. The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ, a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor σ ³². Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 °C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ. Cellular DnaA308 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ-independent manner. Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins.

DOI： 10.1111/j.1365-2443.2004.00800.x

Event date： 2023.6

Language：English   Presentation type：Oral presentation (general)  

Venue：Institute for Integrative Biology of the Cell   Country：France  

Event date： 2023.6

Language：English  

Venue：Institute for Integrative Biology of the Cell   Country：France  

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Event date： 2022.10

Language：English  

Country：Japan  

Event date： 2022.10

Language：English  

Country：Japan  

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Event date： 2022.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2022.3

Language：Japanese  

Venue：オンライン   Country：Japan  

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Event date： 2021.12

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：横浜市   Country：Japan  

Event date： 2021.9

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：東京都（オンライン）   Country：Japan  

複製ヘリカーゼを染色体DNAに装着することは複製開始複合体のミッションである。大腸菌の場合、開始複合体は複製起点上で重合するDnaAタンパク質を核とする。この複合体はダイナミックな構造変化を伴って複製起点の２本鎖DNAを局所的に弛緩し、生じた１本鎖DNAにDnaBヘリカーゼを装着する。この反応はDnaA-DnaB間の直接相互作用を必要とするが、その詳細な分子機構は不明である。我々は大腸菌やカウロバクター菌をモデルとし、試験管内再構成系や変異体解析などを組み合わせることで、新たなDnaBヘリカーゼ装着機構を明らかにした。本発表では、真正細菌の複製ヘリカーゼ装着機構の基本原理について考察したい。

Event date： 2020.4

Language：English   Presentation type：Oral presentation (general)  

Venue：インターネット   Country：United States  

Event date： 2020.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋市   Country：Japan  

細菌染色体の細胞内配置は細胞周期進行と連動してダイナミックに制御される。アルファプロテオバクテリアに属するカウロバクターにおいて、染色体の複製起点と複製終点はそれぞれ異なる細胞極に配置されている。染色体複製が始まると、姉妹複製起点の一つは別の細胞極へと移動し、また、複製終点は細胞中央へと移動し、そこで局在化する。この時、細胞の分裂装置は複製終点と共局在している。この共局在は細胞中央での細胞分裂を保証すると考えられるが、染色体複製終点と分裂装置とを直接連結する分子とそのメカニズムは不明である。本研究において、我々はこの局在性に関与する新規タンパク質を探索し、機能未知DNA結合タンパク質ZapTを同定した。まず欠損株や大量発現株の解析より、ZapTは染色体複製終点と分裂装置との共局在だけでなく、正常な細胞分裂装置の形成制御にも重要であることがわかった。次にChIP解析の結果、ZapTは染色体の複製終点近傍に結合することが示唆された。さらに精製蛋白を用いた性状解析より、ZapTは細胞分裂装置の構成因子と直接結合し、高次な複合体を形成しうることがわかった。これらより、ZapTはカウロバクターの複製終点と分裂装置とを直接連結するためのハブ蛋白として機能すると考えられる。プロテオバクテリア門に属する細菌の多くは機能未知のZapTホモログを保持しているため、ZapTを介した複製終点と分裂装置の制御は細菌界でよく保存された原理かもしれない。

Event date： 2020.2

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：名古屋市   Country：Japan  

Event date： 2019.12

Language：English   Presentation type：Oral presentation (general)  

Venue：福岡市   Country：Japan  

The bacterial chromosomes are spatially and timely organized to execute chromoso
me replication and segregation in a manner coupled to cell division. In the gram
-negative, alphaproteobacterium Caulobacter crescentus, subcellular posit
ioning of the individual chromosomal loci alters dynamically during chromosome r
eplication. In a pre-replication stage, the origin of chromosome replication is
sequestered to one pole of the cell while the terminus region is localized at th
e other cell pole. Upon initiation of chromosome replication, one of the newly-r
eplicated origin DNAs is driven toward the other cell pole to establish bipolar
localization of the origin DNAs. Concomitantly, the terminus region is recruited
to the midcell position where the bacterial tubulin homolog FtsZ colocalizes to
form cytokinetic Z-rings. Although this colocalization likely coordinates cell
division with dynamic chromosome architecture, the molecular basis for a physica
l link between the Z-rings and the terminus region remains unknown. In this stud
y, we identify the terminus-interactive DNA binding protein ZapT and show that i
t directly binds to a component associated with the Z-rings. Moreover, the #Iza
pT#IR gene is crucial to maintain normal cell division control in C. crescentus
. Together, we propose that ZapT acts as a molecular bridge that physically link
s the terminus region to the Z-rings, thereby ensuring precise processes in chro
mosome segregation and cell division. Because ZapT orthologs are conserved among
diverse proteobacterial species, our findings may represent a general mechanism
to coordinate cell division with chromosome organization in time and space.

Event date： 2018.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：横浜国際会議場   Country：Japan  

Event date： 2018.9

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：奈良先端大学   Country：Japan  

Event date： 2018.9

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：岡山大学   Country：Japan  

Event date： 2018.6 - 2018.7

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：九州大学   Country：Japan  

Event date： 2010.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸市   Country：Japan  

Event date： 2010.6

Venue：Freiburg   Country：Germany  

Event date： 2010.6 - 2010.5

Venue：熊本県阿蘇郡南阿蘇村   Country：Japan  

Event date： 2010.5

Venue：長野県南木曽   Country：Japan  

Event date： 2009.11

Venue：滋賀県彦根市   Country：Japan  

Event date： 2009.10

Venue：神戸市   Country：Japan  

Analysis on the minimal functional structure of the DnaA complex for the regulation of duplex DNA unwinding

Event date： 2009.7

Country：Canada  

Event date： 2008.10

Presentation type：Symposium, workshop panel (public)  

Venue：Shizuoka   Country：Japan  

Event date： 2008.9

Presentation type：Symposium, workshop panel (public)  

Venue：名古屋市   Country：Japan  

Analysis on the minimal functional elements of the replication origin in duplex DNA unwinding by DnaA

Presentation type：Oral presentation (general)  

Venue：静岡市   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：Kyoto   Country：United Kingdom  

Presentation type：Oral presentation (general)  

Venue：つくば市   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：熱海   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：名古屋市   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：岡山市   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：Cirencester   Country：United Kingdom  

Event date： 2023.6

Language：Japanese  

Venue：山形県鶴岡市   Country：Japan  

Event date： 2023.6

Language：Japanese  

Venue：山形県鶴岡市   Country：Japan  

Event date： 2023.6

Language：Japanese  

Venue：山形県鶴岡市   Country：Japan  

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Event date： 2023.6

Language：Japanese  

Venue：山形県鶴岡市   Country：Japan  

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Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2022.11

Language：Japanese  

Venue：オンライン   Country：Japan  

researchmap

Event date： 2022.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2022.6

Language：Japanese  

Venue：オンライン   Country：Japan  

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Event date： 2021.9 - 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：富山   Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：富山   Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：富山   Country：Japan  

Event date： 2021.6

Language：Japanese  

Venue：富山   Country：Japan  

researchmap

Event date： 2021.6

Language：Japanese  

Venue：富山   Country：Japan  

researchmap

Event date： 2021.6

Language：Japanese  

Venue：富山   Country：Japan  

researchmap

Event date： 2021.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2020.9

Language：Japanese  

Venue：オンライン   Country：Japan  

Event date： 2019.9

Language：Japanese  

Venue：横浜市   Country：Japan  

Event date： 2019.9

Language：English  

Venue：横浜市   Country：Japan  

Event date： 2019.9

Language：Japanese  

Venue：福井県福井市   Country：Japan  

Event date： 2019.6

Language：Japanese  

Venue：長崎県長崎市   Country：Japan  

Event date： 2019.6

Language：Japanese  

Venue：長崎県長崎市   Country：Japan  

Event date： 2019.5

Language：Japanese  

Venue：滋賀県大津市   Country：Japan  

Event date： 2019.3

Language：Japanese  

Venue：東京都八王子   Country：Japan  

Event date： 2018.11

Language：Japanese  

Venue：横浜国際会議場   Country：Japan  

Event date： 2018.11

Language：English  

Venue：Kanazawa   Country：Japan  

Event date： 2018.11

Language：English  

Venue：Kanazawa   Country：Japan  

Event date： 2018.11

Language：English  

Venue：Kanazawa   Country：Japan  

Event date： 2018.9

Language：Japanese  

Venue：奈良先端大学   Country：Japan  

Event date： 2018.9

Language：English  

Venue：Kyllini   Country：Greece  

Event date： 2018.5

Language：Japanese  

Venue：山形県南陽市   Country：Japan  

Event date： 2018.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：山形県南陽市   Country：Japan  

Event date： 2018.5

Language：Japanese  

Venue：山形県南陽市   Country：Japan  

Event date： 2018.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Event date： 2018.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Event date： 2018.3

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2018.3

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2017.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Event date： 2017.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岐阜市   Country：Japan  

Cyclic diguanosine monophosphate (c-di-GMP)はバクテリアに広く保存された低分子シグナル伝達物質である。この低分子化合物は細胞内の複数の受容体蛋白と結合し、バクテリア細胞のふるまいをグローバルに調節する。有柄真正細菌のカウロバクターでは、c-di-GMP濃度が細胞周期依存的に変動し、G1期からS期にかけて一過的に上昇する。c-di-GMPを合成できないカウロバクター変異株は細胞分裂やゲノム複製などさまざまな細胞活動に異常を起こすため、c-di-GMPはこれらの細胞活動に極めて重要な働きを担うことが示唆される。しかしながら、c-di-GMPの細胞周期制御における分子機構は不明である。今回、我々は二機能性キナーゼ・ホスファターゼとして働く必須蛋白CckAがc-di-GMPと直接結合することを見出した(Nature 2015)。アポ型CckAはキナーゼとして働き、複製開始阻害蛋白CtrAをリン酸化する。このリン酸化型CtrAは複製オリジンに結合し、DnaAによる複製開始反応を抑制する。試験管内で再構成されたCckAのリン酸化カスケードにおいて、我々はc-di-GMPがCckAのホスファターゼ活性を亢進し、CtrAを脱リン酸化することを明らかにした。機能構造解析より、c-di-GMPはCckAのATPaseドメインに結合し、ホスファターゼ型の構造を安定化しうることがわかった(Sci. Adv. 2016)。さらに変異体解析より、細胞内c-di-GMP濃度上昇がCckAを介して複製オリジンをタイミングよくライセンス化することが示唆された。これらの結果より、周期的に変動するc-di-GMPが細胞周期オシレーターとしてゲノム複製を制御することがわかった（図１）。バクテリアにおいて、原理的にサイクリンと同様の働きをもつ細胞周期オシレーターの発見は今回が初めてである。CckAホモログはアルファプロテオバクテリア間で高度に保存されており、c-di-GMPによるCckAを介したゲノム複製制御は多くのバクテリアに共通のメカニズムかもしれない。

Event date： 2010.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸市   Country：Japan  

Event date： 2010.12

Venue：神戸市   Country：Japan  

Event date： 2010.9

Venue：北海道   Country：Japan  

Event date： 2010.6

Venue：Freiburg   Country：Germany  

Event date： 2010.5

Venue：長野県南木曽   Country：Japan  

Event date： 2010.1

Venue：Awaji, Hyogo   Country：Japan  

Event date： 2009.12

Venue：福岡市   Country：Japan  

Event date： 2009.12

Venue：横浜市   Country：Japan  

Mechanism of the inter-DnaA interactions for the ATP-DnaA-dependent activation of initiation complex

Event date： 2009.6

Venue：熱海市   Country：Japan  

Event date： 2008.12

Presentation type：Oral presentation (general)  

Venue：神戸市   Country：Japan  

Analysis on the subcellular localization of DnaA associating protein, DiaA for stimulation of initiation complex formation in E. coli

Event date： 2008.12

Presentation type：Oral presentation (general)  

Venue：神戸市   Country：Japan  

Biochemical analysis for mutants of DnaC, a DNA helicase loader of replication initiation in E.coli

Event date： 2008.12

Presentation type：Oral presentation (general)  

Venue：神戸市   Country：Japan  

Analysis on role for clamp binding of RisA in the chromosome partition in E. coli

Event date： 2008.11

Presentation type：Oral presentation (general)  

Venue：Fukuoka   Country：Japan  

Event date： 2008.9

Venue：名古屋市   Country：Japan  

Event date： 2008.7

Venue：静岡県藤枝市   Country：Japan  

Event date： 2008.7

Venue：静岡県藤枝市   Country：Japan  

Event date： 2008.6

Presentation type：Oral presentation (general)  

Venue：Geilo   Country：Norway  

Event date： 2008.6

Presentation type：Oral presentation (general)  

Venue：東京都江戸川区   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：Santa Fe   Country：United States  

Presentation type：Oral presentation (general)  

Venue：Southern Styria   Country：Austria  

Presentation type：Oral presentation (general)  

Venue：Cirencester   Country：United Kingdom  

Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：Santa Fe   Country：United States  

Presentation type：Oral presentation (general)  

Country：Japan  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publisher：日本ゲノム微生物学会  

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

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Type：Peer review 

Number of peer-reviewed articles in foreign language journals：5

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

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Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

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Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

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Type：Academic society, research group, etc. 

Type：Peer review 

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Type：Peer review 

Number of peer-reviewed articles in foreign language journals：4

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Number of participants：400

Type：Competition, symposium, etc. 

Number of participants：100

Type：Competition, symposium, etc. 

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：1

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

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Type：Competition, symposium, etc. 

Type：Competition, symposium, etc.