ゲノム複製開始点の2重鎖DNAを特異的に開裂する蛋白質高次複合体のメカニズム
キーワード：ゲノム複製、細胞周期、DnaA蛋白質、AAA+ファミリー
2005.10.

主要著書

主要原著論文

1.	Chuyuan Lu, Ryusei Yoshida, Tsutomu Katayama, Shogo Ozaki, Thermotoga maritima oriC involves a DNA unwinding element with distinct modules and a DnaA-oligomerizing region with a novel directional binding mode, Journal of Biological Chemistry, 10.1016/j.jbc.2023.104888, 104888-104888, 2023.07, 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 DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, bacterial 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 initiator (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 single-stranded DUE 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 DNA was also responsible for unwinding. These findings indicate that a single-stranded DUE recruitment mechanism was responsible for unwinding, and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria..
2.	Shogo Ozaki, Dengyu Wang, Yasutaka Wakasugi, Naoto Itani, Tsutomu Katayama, The Caulobacter crescentus DciA promotes chromosome replication through topological loading of the DnaB replicative helicase at replication forks, Nucleic Acids Research, 10.1093/nar/gkac1146, 2022.12, Abstract The replicative DNA helicase translocates on single-stranded DNA to drive replication forks during chromosome replication. In most bacteria the ubiquitous replicative helicase, DnaB, co-evolved with the accessory subunit DciA, but how they function remains incompletely understood. Here, using the model bacterium Caulobacter crescentus, we demonstrate that DciA plays a prominent role in DNA replication fork maintenance. Cell cycle analyses using a synchronized Caulobacter cell population showed that cells devoid of DciA exhibit a severe delay in fork progression. Biochemical characterization revealed that the DnaB helicase in its default state forms a hexamer that inhibits self-loading onto single-stranded DNA. We found that upon binding to DciA, the DnaB hexamer undergoes conformational changes required for encircling single-stranded DNA, thereby establishing the replication fork. Further investigation of the functional structure of DciA revealed that the C-terminus of DciA includes conserved leucine residues responsible for DnaB binding and is essential for DciA in vivo functions. We propose that DciA stimulates loading of DnaB onto single strands through topological isomerization of the DnaB structure, thereby ensuring fork progression. Given that the DnaB-DciA modules are widespread among eubacterial species, our findings suggest that a common mechanism underlies chromosome replication..
3.	Shogo Ozaki, Yasutaka Wakasugi, Tsutomu Katayama, Z-Ring-Associated Proteins Regulate Clustering of the Replication Terminus-Binding Protein ZapT in Caulobacter crescentus, mBio, 10.1128/mBio.02196-20, 2021.01.
4.	Shogo Ozaki, Urs Jenal, Tsutomu Katayama, Novel divisome-associated protein spatially coupling the z-ring with the chromosomal replication terminus in caulobacter crescentus, mBio, 10.1128/mBio.00487-20, 11, 2, 2020.03, [URL], Cell division requires proper spatial coordination with the chromosome, which undergoes dynamic changes during chromosome replication and segregation. FtsZ is a bacterial cytoskeletal protein that assembles into the Z-ring, providing a platform to build the cell division apparatus. In the model bacterium Caulobacter crescentus, the cellular localization of the Z-ring is controlled during the cell cycle in a chromosome replication-coupled manner. Although dynamic localization of the Z-ring at midcell is driven primarily by the replication origin-associated FtsZ inhibitor MipZ, the mechanism ensuring accurate positioning of the Z-ring remains unclear. In this study, we showed that the Z-ring colocalizes with the replication terminus region, located opposite the origin, throughout most of the C. crescentus cell cycle. Spatial organization of the two is mediated by ZapT, a previously uncharacterized protein that inter-acts with the terminus region and associates with ZapA and ZauP, both of which are part of the incipient division apparatus. While the Z-ring and the terminus region coin-cided with the presence of ZapT, colocalization of the two was perturbed in cells lacking zapT, which is accompanied by delayed midcellular positioning of the Z-ring. Moreover, cells overexpressing ZapT showed compromised positioning of the Z-ring and MipZ. These findings underscore the important role of ZapT in controlling cell division pro-cesses. We propose that ZapT acts as a molecular bridge that physically links the terminus region to the Z-ring, thereby ensuring accurate site selection for the Z-ring. Because ZapT is conserved in proteobacteria, these findings may define a general mechanism coordinating cell division with chromosome organization. IMPORTANCE Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. In enteric bacteria, ZapA associates with the cytoskeletal Z-ring and establishes a physical linkage to the chromosomal replication terminus through its interaction with ZapB-MatP-DNA complexes. However, because ZapB and MatP are found only in enteric bacteria, it remains unclear how the Z-ring and the terminus are coordinated in the vast majority of bacteria. Here, we provide evidence that a novel conserved protein, termed ZapT, mediates colocalization of the Z-ring with the terminus in Caulobacter crescentus, a model organism that is phylo-genetically distant from enteric bacteria. Given that ZapT facilitates cell division processes in C. crescentus, this study highlights the universal importance of the physical linkage between the Z-ring and the terminus in maintaining cell integrity..

主要総説, 論評, 解説, 書評, 報告書等

1.	Katayama, T, Ozaki, S, Keyamura, K, Fujimitsu, K., Regulation of the replication cycle: conserved and diverse regulatory systems for DnaA and oriC, Nat Rev Microbiol, 2010, 2010.03.
2.	Ozaki S., Katayama T., DnaA structure, function, and dynamics in the initiation at the chromosomal origin., Plasmid, 2009.07.
3.	尾崎省吾, DnaAによる2重鎖開裂における複製起点の最小機能構造, 日本遺伝学会第80回大会Best Papers賞記念総説, 2008.12.
4.	尾崎省吾, 染色体複製開始点の2重鎖DNA開裂を制御するDnaA新奇機能構造の解析, 日本遺伝学会第79回大会Best Papers賞記念総説, 2007.11.

主要学会発表等

1.	Shogo Ozaki, Chuyuan Lu, Ryusei Yoshida, Yasutaka Wakasugi, Shohei Sato, Tsutomu Katayama, A common mechanism and architecture of the dynamic nucleoprotein complex formed at the origin DNA of eubacterial chromosome replication, 第46回日本分子生物学会年会, 2023.12, Initiation of chromosome replication requires dynamic nucleoprotein complexes to establish replication forks. In Escherichia coli, the origin, oriC, comprises an AT-rich DNA unwinding element (DUE) and asymmetric DnaA box sequences guiding the DnaA initiator to form a highly ordered complex. This initiation complex promotes DUE unwinding and concomitantly binds the resultant single-stranded DUE in a sequence-specific manner, stabilizing the unwound state and facilitating assembly of the replisome components. While DnaA homologs are ubiquitous in eubacteria, the origin sequences differ significantly in respect with the number, direction, and spatial arrangement of DnaA boxes and DUE sequences, limiting our understanding of the general principles governing the process of DUE unwinding. Here, we investigate molecular mechanisms of a prototypic DnaA-oriC complex in the evolutionarily ancient hyperthermophile Thermotoga maritima. We reveal that the DnaA protein from this bacterium retains the ability to form an initiation complex on the cognate oriC, irrespective of DnaA box directions. This flexibility in DnaA assembly is likely driven by substantial swiveling of the DnaA box-binding domain relative to the DnaA AAA+ domain supporting DnaA-DnaA interaction. The resultant DnaA-oriC complexes bind to the three direct repeats of the trinucleotide TAG within single-stranded DUE, stabilizing DUE unwinding (Lu et al. JBC 2023). These findings provide insights into the conservation of fundamental mechanisms for DUE unwinding and single-stranded DUE binding by DnaA proteins across eubacteria, supporting the tunable nature of origin sequences in constructing the initiation complex. .
2.	Shogo Ozaki,Chuyuan Lu, Ryusei Yoshida, Yasutaka Wakasugi, Tsutomu Katayama, A common insight into mechanisms of duplex unwinding at the origin DNA during eubacterial chromosome replication , 第96回日本生化学会大会ワークショップ「染色体DNA複製開始複合体と開始制御メカニズムの新たな展望」, 2023.11.
3.	Shogo Ozaki, The mechanism of chromosome replication in the alpha-proteobacterium Caulobacter crescentus., Research seminar, 2023.06.
4.	Shogo Ozaki, Dengyu Wang, Yasutaka Wakasugi, Naoto Itani, and Tsutomu Katayama, Analysis on the loading mechanism of the bacterial replicative DnaB helicase in the alpha-proteobacterium Caulobacter crescentus., NIG International Symposium 2022, 2022.10.
5.	尾崎　省吾, 井谷直人, 荒田美悠, 片山　勉, プロテオバクテリア綱カウロバクター・クレセンタスにおけるDNA複製ヘリカーゼ装着機構の解析, 第16回日本ゲノム微生物学会年会, 2022.03.
6.	Shogo Ozaki,Tsutomu Katayama, Analysis of the chromosomal replication mechanism in the eubacterium Caulobacter crescentus, 日本分子生物学会第44回大会, 2021.12.
7.	尾﨑省吾,林千尋,井谷直人,吉田竜星,盧楚元,鶴田匠,若杉泰敬,片山勉, 真正細菌の染色体複製における複製ヘリカーゼ装着機構の解析, 日本遺伝学会第93回大会, 2021.09, 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。.
8.	尾崎省吾、若杉泰敬、片山勉, 染色体複製終点と細胞分裂装置とを連係する新規DNA結合タンパク質の解析, 第14回日本ゲノム微生物学会年会, 2020.03, 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。.
9.	Shogo Ozaki、Yasutaka Wakasugi、Urs Jenal, Tsutomu Katayama, A novel divisome-associated protein spatially couples the Z-ring with the chromosomal replication terminus in Caulobacter crescentus, CAULOCONFERENCE 2020, 2020.04.
10.	尾崎省吾、若杉泰敬、片山勉, カウロバクター菌における染色体複製と細胞分裂とを連係する機構の解析, 第93回日本細菌学会総会, 2020.02.
11.	尾崎 省吾, 若杉泰敬, 片山 勉, Identification and characterization of a novel DNA binding protein that colocalizes the chromosome replication terminus with cell division apparatus in Caulobacter crescentus, 第４２回日本分子生物学会年会, 2019.12, 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 za pT 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..
12.	Shogo Ozaki, Urs Jenal, Yasutaka Wakasugi, Tsutomu Katayama, The role of the bacterial second messenger signaling for chromosome segregation dynamics in Caulobacter crescentus, 第４１回日本分子生物学会年会, 2018.11.
13.	Shogo Ozaki, Christian Lori, Badri N Dubey, Urs Jenal, The role of the second messenger signalling for coordinating chromosome replication in time and space., 日本遺伝学会第９０回大会, 2018.09.
14.	Shogo Ozaki, Lori Christian, Urs Jenal, The second messenger signaling drives chromosome replication in the asymmetrically dividing bacterium Caulobacter crescentus, 第５６回日本生物物理学会年会, 2018.09.
15.	Shogo Ozaki, Lori Christian, Urs Jenal, The role for the bacterial second-messenger during the bacterial chromosome replication, 平成３０年度日本生化学会九州支部例会, 2018.06.
16.	尾崎省吾、片山　勉, Functional organization of the highly-ordered DnaA-oriC complex for the initiation of chromosomal replication in Escherichia coli, 第33回日本分子生物学会年会　第83回日本生化学会大会　合同大会, 2010.12.
17.	Shogo Ozaki, Tsutomu Katayama, Mechanism for ATP-DnaA-dependent unwinding of duplex DNA within the chromosomal replication origin, EMBO Workshop on Replication & Segregation of Chromosomes, 2010.06.
18.	尾崎省吾、片山　勉, 大腸菌染色体複製開始を制御するDnaA-oriC複合体の機能構造, 第7回21世紀大腸菌研究会, 2010.06.
19.	尾崎省吾、毛谷村賢司、松田雄作、片山　勉, 複製装置クランプサブユニットと結合する新規蛋白CrfC を介した染色体分配機構の解析, 第8回大腸菌研究会, 2010.05.
20.	尾崎省吾、片山勉, DnaAによる2本鎖開裂を制御するための複製開始点の新規機能構造特性, 第20回DNA複製・組換え・修復ワークショップ, 2009.11.
21.	尾崎省吾、片山勉, 複製開始点の2本鎖開裂を制御するDnaA複合体の最小機能構造, 日本生化学会第82回大会, 2009.10.
22.	Shogo Ozaki, Tsutomu Katayama, Specific interaction between DnaA AAA+ domain and the single-stranded DNA promotes duplex DNA unwinding during the initiation of chromosomal replication, 8th Inthernational Meeting on AAA Proteins, 2009.07.
23.	Shogo Ozaki, Kenta Nakamura and Tsutomu Katayama, A common role of the DUE binding activity of DnaA for the initiation of chromosomal replication, 3R symposium 2008, 2008.10.
24.	尾崎省吾、片山　勉, DnaAによる2重鎖開裂における複製起点の最小機能構造, 日本遺伝学会第80回大会, 2008.09.
25.	尾崎省吾、川上広宣、中村賢太、藤川乃り映、香川亘、朴　三用、横山茂之、胡桃坂仁志、片山勉, 複製開始複合体中で特異的に形成されるDnaA機能領域を介した複製開始点開裂の分子機構, 組換え・複製合同ワークショップ, 2008.03.
26.	Ozaki, S, Kawakami, H, Nakamura, K, Fujikawa, N, Kagawa, W, Yokoyama, S, Kurumizaka, H and Katayama, T., Role for specific residues of DnaA AAA+ domain in duplex DNA unwinding during the initiation of chromosomal replication, 7th International conference on AAA+ proteins, 2007.09.
27.	尾崎省吾、川上広宣、中村賢太、藤川乃り映、香川　亘、横山茂之、胡桃坂仁志、片山　勉, 染色体複製開始点の2重鎖DNA開裂を制御するDnaA新奇機能構造の解析, 日本遺伝学会第79回大会, 2007.09.
28.	尾崎省吾、川上広宣、鈴木滋夫、中村賢太、胡桃坂仁志、片山勉, DnaA蛋白質の特異的AAA+領域を介した複製開始点開裂のメカニズム, 分子生物学会シンポジウム, 2006.12.
29.	尾崎省吾、川上広宣、鈴木滋夫、中村賢太、千里内啓行、末次正幸、藤光和之、片山勉, DnaAのAAA+ sensor 1モチーフを介した染色体複製開始の制御機構, DNA複製ワークショップ, 2006.11.
30.	尾崎省吾、川上広宣、鈴木滋夫、中村賢太、千里内啓行、末次正幸、藤光和之、片山勉, 大腸菌複製開始蛋白質DnaAにおけるAAA+ sensor 1モチーフの特異的な機能置換, 日本遺伝学会第78回大会, 2006.09.
31.	Ozaki, S, Kurumizaka, H and Katayama, T., ATP-dependent activation of an initiation complex in the minichromosomal replication of hyperthermophile Thermotoga maritima in vitro, 20th IUBMB International Congress of Biochemistry & Molecular Biology , 2006.06.

所属学会名

学協会役員等への就任

学会大会・会議・シンポジウム等における役割

学会誌・雑誌・著書の編集への参加状況

学術論文等の審査

海外渡航状況, 海外での教育研究歴

外国人研究者等の受入れ状況

研究奨励賞, 日本ゲノム微生物学会, 2023.03.

平成31年度科学技術分野の文部科学大臣表彰 若手科学者賞, 文部科学省, 2019.04.

若手優秀発表賞, 第２４回DNA複製・組換え・修復ワークショップ運営委員会, 2017.11.

平成２８年度日本分子生物学会 富澤純一・桂子 基金 若手賞, 日本分子生物学会, 2016.05.

平成23年 上原記念生命科学財団ポストドクトラルフェロー, 上原記念生命科学財団, 2011.03.

平成23年 持田記念医学薬学振興財団留学助成, 持田記念医学薬学振興財団, 2011.10.

日本遺伝学会第80回大会Best Papers賞, 日本遺伝学会, 2008.11.

日本遺伝学会第79回大会Best Papers賞, 日本遺伝学会, 2007.11.