Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nar/gkz795

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

DOI： 10.1074/jbc.M116.762815

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/gtc.12395

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase.

DOI： 10.1093/nar/gku1051

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

The initiation of chromosomal DNA replication is rigidly regulated to ensure that it occurs in a cell cycle-coordinated manner. To ensure this in Escherichia coli, multiple systems regulate the activity of the replication initiator ATP-DnaA. The level of ATP-DnaA increases before initiation after which it drops via DnaA-ATP hydrolysis, yielding initiation-inactive ADP-DnaA. DnaA-ATP hydrolysis is crucial to regulation of initiation and mainly occurs by a replication-coupled feedback mechanism named RIDA (regulatory inactivation of DnaA). Here, we report a second DnaA-ATP hydrolysis system that occurs at the chromosomal site datA. This locus has been annotated as a reservoir for DnaA that binds many DnaA molecules in a manner dependent upon the nucleoid-associated factor IHF (integration host factor), resulting in repression of untimely initiations; however, there is no direct evidence for the binding of many DnaA molecules at this locus. We reveal that a complex consisting of datA and IHF promotes DnaA-ATP hydrolysis in a manner dependent on specific inter-DnaA interactions. Deletion of datA or the ihf gene increased ATP-DnaA levels to the maximal attainable levels in RIDA-defective cells. Cell-cycle analysis suggested that IHF binds to datA just after replication initiation at a time when RIDA is activated. We propose a model in which cell cycle-coordinated ATP-DnaA inactivation is regulated in a concerted manner by RIDA and datA.

DOI： 10.1073/pnas.1212070110

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

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

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

Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.

DOI： 10.1016/j.bbamcr.2021.119167

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

PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3'-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.

DOI： 10.1093/nar/gkab634

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

The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

DOI： 10.1021/jacs.9b11232

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

DOI： 10.3389/fmicb.2019.00072

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

In Escherichia coli, the level of the ATP-DnaA initiator is increased temporarily at the time of replication initiation. The replication origin, oriC, contains a duplex-unwinding element (DUE) flanking a DnaA-oligomerization region (DOR), which includes twelve DnaA-binding sites (DnaA boxes) and the DNA-bending protein IHF-binding site (IBS). Although complexes of IHF and ATP-DnaA assembly on the DOR unwind the DUE, the configuration of the crucial nucleoprotein complexes remains elusive. To resolve this, we analyzed individual DnaA protomers in the complex and here demonstrate that the DUE-DnaA-box-R1-IBS-DnaA-box-R5M region is essential for DUE unwinding. R5M-bound ATP-DnaA predominantly promotes ATP-DnaA assembly on the DUE-proximal DOR, and R1-bound DnaA has a supporting role. This mechanism might support timely assembly of ATP-DnaA on oriC. DnaA protomers bound to R1 and R5M directly bind to the unwound DUE strand, which is crucial in replication initiation. Data from in vivo experiments support these results. We propose that the DnaA assembly on the IHF-bent DOR directly binds to the unwound DUE strand, and timely formation of this ternary complex regulates replication initiation. Structural features of oriC support the idea that these mechanisms for DUE unwinding are fundamentally conserved in various bacterial species including pathogens.

DOI： 10.1093/nar/gkx914

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

DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA.

DOI： 10.1111/1462-2920.12147

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

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

Event date： 2023.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：九州大学   Country：Japan  

Event date： 2022.11 - 2022.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：幕張メッセ   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：静岡県三島市   Country：Japan  

Event date： 2021.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：パシフィコ横浜   Country：Japan  

Event date： 2021.11

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2021.10

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2016.11

Language：English   Presentation type：Oral presentation (general)  

Venue：Hotel Ichibata, Matsue, Shimane   Country：Japan  

Event date： 2016.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：日本大学国際関係学部三島駅北口校舎   Country：Japan  

Event date： 2016.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島大学郡元キャンパス   Country：Japan  

Event date： 2015.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸コンベンションセンター   Country：Japan  

Event date： 2015.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：静岡県焼津市   Country：Japan  

Event date： 2014.11

Language：English  

Venue：Shizuoka   Country：Japan  

Event date： 2014.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：都市センターホテル 5 階 オリオン   Country：Japan  

Event date： 2014.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長浜バイオ大学   Country：Japan  

Event date： 2014.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：九州大学病院キャンパス コラボステーション I   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：慶應大学日吉キャンパス   Country：Japan  

Venue：神戸   Country：Japan  

Event date： 2016.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京工業大学 大岡山キャンパス   Country：Japan  

Event date： 2015.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸コンベンションセンター   Country：Japan  

Event date： 2015.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸コンベンションセンター   Country：Japan  

Event date： 2015.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：焼津グランドホテル、静岡県焼津市   Country：Japan  

Event date： 2015.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：静岡県焼津市   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：佐賀大学   Country：Japan  

Venue：名古屋大学   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：修善寺   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：慶應大学日吉キャンパス   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：慶應大学日吉キャンパス   Country：Japan  

Venue：横浜   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Venue：神戸   Country：Japan  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：千葉大学   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Language：Japanese  

Country：Japan  

Language：Japanese  

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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.