九州大学-研究者情報 [河添好孝 (助教) 理学研究院生物科学部門]

1.	Yoshitaka Kawasoe, Toshiki Tsurimoto, Takuro Nakagawa, Hisao Masukata, Tatsuro S. Takahashi, MutSα maintains the mismatch repair capability by inhibiting PCNA unloading., eLife, 10.7554/eLife.15155, 5, 2016JULY, 2016.07, [URL], Eukaryotic mismatch repair (MMR) utilizes single-strand breaks as signals to target the strand to be repaired. DNA-bound PCNA is also presumed to direct MMR. The MMR capability must be limited to a post-replicative temporal window during which the signals are available. However, both identity of the signal(s) involved in the retention of this temporal window and the mechanism that maintains the MMR capability after DNA synthesis remain unclear. Using Xenopus egg extracts, we discovered a mechanism that ensures long-term retention of the MMR capability. We show that DNA-bound PCNA induces strand-specific MMR in the absence of strand discontinuities. Strikingly, MutS a inhibited PCNA unloading through its PCNA-interacting motif, thereby extending significantly the temporal window permissive to strand-specific MMR. Our data identify DNA-bound PCNA as the signal that enables strand discrimination after the disappearance of strand discontinuities, and uncover a novel role of MutS a in the retention of the post-replicative MMR capability..
2.	Atsuhiro Shimada, Yoshitaka Kawasoe, Yoshito Hata, Tatsuro S. Takahashi, Ryoji Masui, Seiki Kuramitsu, Kenji Fukui, MutS stimulates the endonuclease activity of MutL in an ATP-hydrolysis-dependent manner, FEBS JOURNAL, 10.1111/febs.12344, 280, 14, 3467-3479, 2013.07, [URL], In the initial steps of DNA mismatch repair, MutS recognizes a mismatched base and recruits the latent endonuclease MutL onto the mismatch-containing DNA in concert with other proteins. MutL then cleaves the error-containing strand to introduce an entry point for the downstream excision reaction. Because MutL has no intrinsic ability to recognize a mismatch and discriminate between newly synthesized and template strands, the endonuclease activity of MutL is strictly regulated by ATP-binding in order to avoid nonspecific degradation of the genomic DNA. However, the activation mechanism for its endonuclease activity remains unclear. In this study, we found that the coexistence of a mismatch, ATP and MutS unlocks the ATP-binding-dependent suppression of MutL endonuclease activity. Interestingly, ATPase-deficient mutants of MutS were unable to activate MutL. Furthermore, wild-type MutS activated ATPase-deficient mutants of MutL less efficiently than wild-type MutL. We concluded that ATP hydrolysis by MutS and MutL is involved in the mismatch-dependent activation of MutL endonuclease activity..
3.	Niyo Kato, Yoshitaka Kawasoe, Hannah Williams, Elena Coates, Upasana Roy, Yuqian Shi, Lorena S. Beese, Orlando D. Scharer, Hong Yan, Max E. Gottesman, Tatsuro S. Takahashi, Jean Gautier, Sensing and Processing of DNA Interstrand Crosslinks by the Mismatch Repair Pathway, CELL REPORTS, 10.1016/j.celrep.2017.10.032, 21, 5, 1375-1385, 2017.10, [URL], DNA interstrand crosslinks (ICLs) that are repaired in non-dividing cells must be recognized independently of replication-associated DNA unwinding. Using cell-free extracts from Xenopus eggs that support neither replication nor transcription, we establish that ICLs are recognized and processed by the mismatch repair (MMR) machinery. We find that ICL repair requires MutS alpha (MSH2-MSH6) and the mismatch recognition FXE motif in MSH6, strongly suggesting that MutS alpha functions as an ICL sensor. MutS alpha recruits MutL alpha and EXO1 to ICL lesions, and the catalytic activity of both these nucleases is essential for ICL repair. As anticipated for a DNA unwinding-independent recognition process, we demonstrate that least distorting ICLs fail to be recognized and repaired by the MMR machinery. This establishes that ICL structure is a critical determinant of repair efficiency outside of DNA replication..
4.	Riki Terui, Koji Nagao, Yoshitaka Kawasoe, Kanae Taki, Torahiko L. Higashi, Seiji Tanaka, Takuro Nakagawa, Chikashi Obuse, Hisao Masukata, Tatsuro S. Takahashi, Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1, Genes and Development, 10.1101/gad.310995.117, 32, 11-12, 806-821, 2018.06, [URL], Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in Xenopus egg extracts. The exclusion was dependent on the Msh2–Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of FUN30, the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with MSH6 or MSH3 deletion but no significant increase with MSH2 deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction..
5.	Mathew J K Jones, Camille Gelot, Stephanie Munk, Amnon Koren, Yoshitaka Kawasoe, Kelly A George, Ruth E Santos, Jesper V Olsen, Steven A McCarroll, Mark G Frattini, Tatsuro S Takahashi, Prasad V Jallepalli, Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication., Molecular cell, 10.1016/j.molcel.2021.01.004, 81, 3, 426-441, 2021.02, [URL], Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes..
6.	Ryota Miyashita, Atsuya Nishiyama, Weihua Qin, Yoshie Chiba, Satomi Kori, Norie Kato, Chieko Konishi, Soichiro Kumamoto, Hiroko Kozuka-Hata, Masaaki Oyama, Yoshitaka Kawasoe, Toshiki Tsurimoto, Tatsuro S Takahashi, Heinrich Leonhardt, Kyohei Arita, Makoto Nakanishi, The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5, eLife, https://doi.org/10.7554/eLife.79013, 12, e79013, 2023.02, [URL], UHRF1-dependent ubiquitin signaling plays an integral role in the regulation of maintenance DNA methylation. UHRF1 catalyzes transient dual mono-ubiquitylation of PAF15 (PAF15Ub2), which regulates the localization and activation of DNMT1 at DNA methylation sites during DNA replication. Although the initiation of UHRF1-mediated PAF15 ubiquitin signaling has been relatively well characterized, the mechanisms underlying its termination and how they are coordinated with the completion of maintenance DNA methylation have not yet been clarified. This study shows that deubiquitylation by USP7 and unloading by ATAD5 (ELG1 in yeast) are pivotal processes for the removal of PAF15 from chromatin. On replicating chromatin, USP7 specifically interacts with PAF15Ub2 in a complex with DNMT1. USP7 depletion or inhibition of the interaction between USP7 and PAF15 results in abnormal accumulation of PAF15Ub2 on chromatin. Furthermore, we also find that the non-ubiquitylated form of PAF15 (PAF15Ub0) is removed from chromatin in an ATAD5-dependent manner. PAF15Ub2 was retained at high levels on chromatin when the catalytic activity of DNMT1 was inhibited, suggesting that the completion of maintenance DNA methylation is essential for the termination of UHRF1-mediated ubiquitin signaling. This finding provides a molecular understanding of how the maintenance DNA methylation machinery is disassembled at the end of the S phase..

主要総説, 論評, 解説, 書評, 報告書等

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1.	河添好孝, 高橋達郎, 放射標識ヌクレオチドを用いた染色体複製・維持研究, 九州大学アイソトープ統合安全管理センター, 2018.10.

主要学会発表等

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1.	河添好孝、織⽥⾥美、坂詰彩、久持涼⼦、宮田太成、高橋達郎, WernerヘリカーゼとMutLαエンドヌクレアーゼは⼀本鎖アニーリングの正確性を制御する, 第27回 DNA複製・組換え・修復ワークショップ, 2023.06.
2.	久持涼⼦、坂詰彩、河添好孝、高橋達郎, 試験管内再構成による抗組換え反応のメカニズムの解析, 第45回日本分子生物学会年会, 2022.11, [URL].
3.	河添好孝、織⽥⾥美、坂詰彩、久持涼⼦、高橋達郎, WernerヘリカーゼとMutLαエンドヌクレアーゼは⼀本鎖アニーリングの正確性を制御する, 第45回日本分子生物学会年会, 2022.11.
4.	河添好孝、織⽥⾥美、坂詰彩、高橋達郎, WernerヘリカーゼとMutLαエンドヌクレアーゼは⼀本鎖アニーリングの正確性を制御する, 第95回日本生化学会大会, 2022.11, [URL].
5.	河添好孝、坂詰彩、織田里美、高橋達郎, WernerヘリカーゼとMutLαエンドヌクレアーゼはDNA二重鎖切断修復の正確性を制御する, 第44回日本分子生物学会年会, 2021.12.
6.	福田翔太、河添好孝、高橋達郎, クロマチンを基質としたミスマッチ修復反応の試験管内再構成に向けた解析, 第44回日本分子生物学会年会, 2021.12.
7.	河添好孝、織⽥⾥美、坂詰彩、久持涼⼦、高橋達郎, WernerヘリカーゼとMutLαエンドヌクレアーゼは⼀本鎖アニーリングの正確性を制御する, 第26回 DNA複製・組換え・修復ワークショップ, 2021.10.
8.	河添好孝,坂詰彩, 織田里美、高橋達郎, WRNヘリカーゼはDNA二重鎖切断修復の正確性維持に重要である, 第38回染色体ワークショップ・第19回核ダイナミクス研究会, 2021.01.
9.	河添好孝, 下川紗貴子 , 釣本敏樹 , 高橋達郎, ツメガエル卵抽出液におけるPCNAアンローディングは主にElg1-RFCが担う, 第42回日本分子生物学会年会, 2019.12.
10.	河添好孝、下川紗貴子、釣本敏樹、高橋達郎, Elg1‒Rfc2‒5複合体（Elg1‒RFC）はツメガエル卵抽出液における主たるPCNAアンローディング複合体である, 第41回日本分子生物学会年会, 2018.11.
11.	Yoshitaka Kawasoe, Sakiko Shimokawa, Toshiki Tsurimoto and Tatsuro Takahashi, The Elg1-Rfc2–5 complex (Elg1-RFC) is the major PCNA-unloading complex in Xenopus egg extracts, 岡崎フラグメント—不連続DNA 複製モデル５０周年記念国際シンポジウム, 2018.12.
12.	河添　好孝、釣本　敏樹、中川　拓郎、升方　久夫、高橋達郎, ミスマッチ修復因子MutSαはPCNAのDNAからの解離を阻害して修復の鎖特異性を保持する, 第23回DNA複製・組換え・修復ワークショップ, 2015.10.
13.	Yoshitaka Kawasoe, Toshiki Tsurimoto, Takuro Nakagawa, Hisao Masukata and Tatsuro Takahashi, MutSα prevents dissociation of PCNA from DNA to keep strand information for eukaryotic mismatch repair, 3R Symposium, 2014.11.
14.	河添　好孝、釣本　敏樹、中川　拓郎、升方　久夫、高橋達郎, ミスマッチ修復因子MutSαはPCNAのDNAからの解離を阻害することで新生鎖の記憶を保持する, 第37回日本分子生物学会年会, 2014.11.
15.	Yoshitaka Kawasoe, Toshiki Tsurimoto, Takuro Nakagawa, Hisao Masukata and Tatsuro Takahashi, Evidence that DNA-loaded PCNA acts as the strand marker for eukaryotic mismatch repair, Cold Spring Harbor Laboratory Meeting 2013, EUKARYOTIC DNA REPLICATION & GENOME MAINTENANCE, 2013.09.
16.	河添好孝, Orientation of PCNA loading determines the strand specificity of the eukaryotic mismatch repair reaction, National Tsing Hua University‐Osaka University Life Science Student Activity Fair 2013 Student Symposium, 2013.05.
17.	河添　好孝、釣本　敏樹、中川　拓郎、升方　久夫、高橋達郎, PCNAはミスマッチ修復反応のDNA鎖特異性を決定する, 第35回日本分子生物学会年会, 2012.12.
18.	河添　好孝、釣本　敏樹、中川　拓郎、升方　久夫、高橋達郎, PCNA plays a key role in identification of the newly synthesized DNA strand in eukaryotic mismatch repair, 第21回 DNA複製・組換え・ゲノム安定性制御ワークショップ, 2011.10.

学会活動

所属学会名

日本生化学会

日本分子生物学会

学会大会・会議・シンポジウム等における役割

2022.11.09～2022.11.11, 第95回日本生化学会大会, オーガナイザー.

2021.01.16～2021.01.21, 第38回染色体ワークショップ・第19回核ダイナミクス研究会, 座長.

学術論文等の審査

年度	外国語雑誌査読論文数	日本語雑誌査読論文数	国際会議録査読論文数	国内会議録査読論文数	合計
2023年度	1	0	0	0	1

受賞

大阪大学理学部賞, 大阪大学, 2011.03.

第21回DNA複製・修復・ゲノム安定性ワークショップ優秀発表賞, 2011.10.

第23回 DNA複製・修復・組換えワークショップ優秀発表賞, 2015.10.

研究資金

科学研究費補助金の採択状況(文部科学省、日本学術振興会)

2022年度～2023年度, 若手研究, 代表, 相同組換えの正確性制御メカニズムの試験管内再構成による解明.

2019年度～2021年度, 若手研究, 代表, ミスマッチ修復機構によるPCNAダイナミクス制御メカニズムの解明.

2017年度～2018年度, 研究活動スタート支援, 代表, PCNAアンローディングをミスマッチ修復機構が制御するメカニズムの解明.

日本学術振興会への採択状況(科学研究費補助金以外)

2013年度～2015年度, 特別研究員, 代表, ミスマッチ修復機構がＤＮＡ複製と協調して働くメカニズムの解明.

競争的資金(受託研究を含む)の採択状況

2023年度～2023年度, 2023年度スカラー共同研究Phase1, 代表, 光照射による特定反応誘導可能な新規試験管内生命現象解析ツールの構築とその評価.

2022年度～2022年度, 2022年度豊田理研スカラー, 代表, 試験管内再構成による相同組換え反応正確性制御メカニズムの解明.

2019年度～2019年度, 2019年度上原記念生命科学財団研究奨励金, 代表, 合成エラー修復／抗組換え反応の分岐機構の生化学解析.

学内資金・基金等への採択状況

2021年度～2022年度, 令和3（2021）年度理学研究院若手支援プロジェクト・プロジェクト支援, 代表, 試験管内再構成によるDNA二重鎖切断修復の正確性を高める反応の制御メカニズムの解明.

九大関連コンテンツ

pure2017年10月2日から、「九州大学研究者情報」を補完するデータベースとして、Elsevier社の「Pure」による研究業績の公開を開始しました。

QIR　九州大学学術情報リポジトリシステム情報科学研究院

理学部・理学研究院


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