・DNAミスマッチ修復の動作原理の解明
・DNAミスマッチ修復とクロマチン形成の協調機構の解明
・相同性依存的修復の正確性を維持する機構の解明
・染色体接着とDNA複製の協調機構の解明
キーワード：DNA複製, DNA修復, 試験管内系, 染色体, ツメガエル
2016.10.

主要原著論文

1.	Yoshitaka Kawasoe, Sakiko Shimokawa, Peter J Gillespie, J Julian Blow, Toshiki Tsurimoto, Tatsuro S Takahashi, The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts., The Journal of Biological Chemistry, 10.1016/j.jbc.2023.105588, 300, 1, 105588-105588, 2024.01, Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors..
2.	Kensuke Tatsukawa, Reihi Sakamoto, Yoshitaka Kawasoe, Yumiko Kubota, Toshiki Tsurimoto, Tatsuro S Takahashi, Eiji Ohashi, Resection of DNA double-strand breaks activates Mre11-Rad50-Nbs1- and Rad9-Hus1-Rad1-dependent mechanisms that redundantly promote ATR checkpoint activation and end processing in Xenopus egg extracts., Nucleic Acids Research, 10.1093/nar/gkae082, 2024.02, Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system..
3.	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 & development, 10.1101/gad.310995.117, 32, 806-821, 2018.06, 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..
4.	Yoshitaka Kawasoe, Toshiki Tsurimoto, Takuro Nakagawa, Hisao Masukata, Tatsuro 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, MutSα 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 MutSα in the retention of the post-replicative MMR capability..
5.	Torahiko L. Higashi, Megumi Ikeda, Hiroshi Tanaka, Takuro Nakagawa, Masashige Bando, Katsuhiko Shirahige, Yumiko Kubota, Haruhiko Takisawa, Hisao Masukata, Tatsuro Takahashi, The prereplication complex recruits XEco2 to chromatin to promote cohesin acetylation in Xenopus egg extracts, Current Biology, 10.1016/j.cub.2012.04.013, 22, 11, 977-988, 2012.06, [URL], Background: Sister chromatids are held together by the ring-shaped cohesin complex, which is loaded onto chromosomes before DNA replication. Cohesion between sister chromosomes is established during DNA replication, and it requires acetylation of the Smc3 subunit of cohesin by evolutionally conserved cohesin acetyltransferases (CoATs). However, how CoATs are recruited to chromatin and how cohesin acetylation is regulated remain unclear. Results: We found that cohesin acetylation requires pre-RC-dependent chromatin loading of cohesin, but surprisingly, it is independent of DNA synthesis in Xenopus egg extracts. Immunodepletion experiments revealed that XEco2 is the CoAT responsible for Smc3 acetylation and sister chromatid cohesion. Recruitment of XEco2 onto chromatin was dependent on pre-RC assembly but was independent of cohesin loading and DNA synthesis. Two short N-terminal motifs, PBM-A and PBM-B, which are conserved among vertebrate Esco2/XEco2 homologs, were collectively essential for pre-RC-dependent chromatin association of XEco2, cohesin acetylation, and subsequent sister chromatid cohesion. The conserved PCNA-interacting protein box in XEco2 was largely dispensable for Smc3 acetylation but was partially required for cohesion. Interaction of acetylated cohesin with DNA was stabilized against salt-wash treatments after DNA replication. Conclusions: Our results demonstrate that pre-RC formation regulates chromatin association of XEco2 in Xenopus egg extracts. We propose that this reaction is critical to acetylate cohesin, whose DNA binding is subsequently stabilized by DNA replication..
6.	Tatsuro Takahashi, Abhijit Basu, Vladimir Bermudez, Jerard Hurwitz, Johannes C. Walter, Cdc7-Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts, Genes and Development, 10.1101/gad.1683308, 22, 14, 1894-1905, 2008.07, [URL], To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2-Scc4 complex, but little is known about how Scc2-Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2-Scc4 exists in a stable complex with the Cdc7-Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2-Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2-Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2-Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion..
7.	Tatsuro Takahashi, Johannes C. Walter, Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication, Genes and Development, 10.1101/gad.1339805, 19, 19, 2295-2300, 2005.10, [URL], Cdc7, a protein kinase required for the initiation of eukaryotic DNA replication, is activated by a regulatory subunit, Dbf4. A second activator of Cdc7 called Drf1 exists in vertebrates, but its function is unknown. Here, we report that in Xenopus egg extracts, Cdc7-Drf1 is far more abundant than Cdc7-Dbf4, and removal of Drf1 but not Dbf4 severely inhibits phosphorylation of Mcm4 and DNA replication. After gastrulation, when the cell cycle acquires somatic characteristics, Drf1 levels decline sharply and Cdc7-Dbf4 becomes the more abundant kinase. These results identify Drf1 as a developmentally regulated, essential activator of Cdc7 in Xenopus..
8.	Tatsuro Takahashi, Pannyun Yiu, Michael F. Chou, Steven Gygi, Johannes C. Walter, Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex, Nature Cell Biology, 10.1038/ncb1177, 6, 10, 991-996, 2004.10, [URL], Cohesin is a multi-subunit, ring-shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase. In yeast, the loading of cohesin onto chromosomes requires the Scc2 protein. In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown. Concurrent with cohesin binding, pre-replication complexes (pre-RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1 and MCM2-7 (the 'licensing' reaction). In S phase, the protein kinase Cdk2 activates pre-RCs, causing origin unwinding and DNA replication. Here, we use Xenopus egg extracts to show that the recruitment of cohesins to chromosomes requires fully licensed chromatin and is dependent on ORC, Cdc6, Cdt1 and MCM2-7, but is independent of Cdk2. We further show that Xenopus Scc2 is required for cohesin loading and that binding of XScc2 to chromatin is MCM2-7 dependent. Our results define a novel pre-RC-dependent pathway for cohesin recruitment to chromosomes in a vertebrate model system..

主要総説, 論評, 解説, 書評, 報告書等

主要学会発表等

1.	Yoshitaka Kawasoe, Satomi Oda, Aya Sakazume, Taisei Miyata, Tatsuro Takahashi, Regulation of the fidelity of homology-directed repair in Xenopus egg extracts, 第96回日本生化学会大会, 2023.10.
2.	Eiichiro Kanatsu, Riki Terui, Yasukazu Daigaku, Tatsuro Takahashi, Mechanistic details of the chromatin remodeling reaction associated with post-replicative DNA mismatch repair, Eukaryotic DNA replication & genome maintenance, 2023.09.
3.	金津　瑛一郎、照井　利輝、高橋　達郎, The mechanism of a chromatin-remodeling reaction associated with replication error correction, 第45回日本分子生物学会年会, 2022.12.
4.	金津　瑛一郎、照井　利輝、高橋　達郎, The mechanism of a chromatin-remodeling reaction associated with replication error correction, Chromosome Replication in the New Era - Old and New Questions in Life Science -, 2022.11.
5.	Tatsuro Takahashi, Yoshitaka Kawasoe, Satomi Oda, Aya Sakazume, Regulation of the fidelity of homology-directed repair in Xenopus egg extracts, The 11th quinquennial conference on DNA repair, 2022.03, [URL], The mismatch repair (MMR) system protects genetic information by handling mispairs arising from DNA replication errors and homology-directed repair between divergent sequences. Replication errors are corrected by the MMR system in a strand-specific manner to restore original genetic information. In contrast, homology-directed repair between divergent sequences is suppressed through the unwinding of intermediates. Genetic studies in yeast have shown that this process, called anti-recombination or heteroduplex rejection, depends on the Msh2-Msh6 (MutSα) mismatch recognition complex and RecQ homolog DNA helicase Sgs1. However, mechanistic details and the regulation of anti-recombination remain still ambiguous, especially in vertebrates, partly due to the insufficiency of in vitro model systems. In this study, we set up a single-strand annealing (SSA) model system in Xenopus egg extracts and found that sequence divergence between two repeating units significantly delays the annealing reaction and reduces the efficiency of SSA. Immunodepletion experiments showed that this reduction of SSA is mediated by MutSα and the Werner helicase. MutSα and the Werner helicase were also important for the fidelity of SSA. We will discuss possible mechanisms and regulations of how these factors increase the SSA fidelity..
6.	照井　利輝、金津　瑛一郎、高橋　達郎, MutSαとSmarcad1はミスマッチ依存的ヌクレオソームリモデリング複合体を形成する, 第94回日本生化学会大会, 2021.11.
7.	照井　利輝、金津　瑛一郎、高橋　達郎, ミスマッチ修復に伴うクロマチンリモデリング反応の試験管内再構成による解析, 第93回日本遺伝学会年会, 2021.09.

所属学会名

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