研究実績
2026
3.
Taisei Miyata, Naoki Tani, Yoshitaka Kawasoe, Kei-ichiro Ishiguro, Tatsuro S Takahashi
bioRxiv, 2026.
Abstract | Links | タグ: Takahashi G
@unpublished{Miyata2026,
title = {Replication protein A prevents unregulated priming and Rad51 loading on single-stranded DNA in nuclear extracts of Xenopus eggs},
author = {Taisei Miyata and Naoki Tani and Yoshitaka Kawasoe and Kei-ichiro Ishiguro and Tatsuro S Takahashi},
url = {http://biorxiv.org/lookup/doi/10.64898/2026.06.09.731035},
doi = {10.64898/2026.06.09.731035},
year = {2026},
date = {2026-06-10},
urldate = {2026-06-10},
publisher = {openRxiv},
abstract = {In eukaryotes, single-stranded DNA (ssDNA) generated during DNA replication, recombination, and repair is rapidly bound and protected by the major single-stranded DNA-binding protein replication protein A (RPA). RPA not only stabilizes ssDNA but also acts as a central platform that coordinates diverse DNA transactions. Exhaustion of RPA due to unregulated ssDNA production leads to replication fork breakage and replication catastrophe, underscoring its critical role in genome stability. However, the direct consequences of RPA limitation remain incompletely understood. Using Xenopus egg extracts, we show that excess ssDNA induces spontaneous priming, a reaction that is otherwise prevented in a physiological nuclear environment. We provide evidence that priming suppression is mediated by stoichiometric binding of RPA to ssDNA. Analysis of the ssDNA-binding proteome reveals that RPA promotes the association of ATR checkpoint factors, Polα–primase, and the RFWD3 ubiquitin ligase with ssDNA. In contrast, RPA depletion induces the recruitment of Rad51, Rad51 paralogs, and Fbh1, a DNA helicase that interacts with both RPA and Rad51 and promotes fork breakage under replication stress. Collectively, our findings suggest that RPA contributes to genome stability by protecting ssDNA from inappropriate DNA synthesis and unscheduled recruitment of recombination and fork-processing factors.},
howpublished = {bioRxiv},
keywords = {Takahashi G},
pubstate = {published},
tppubtype = {unpublished}
}
In eukaryotes, single-stranded DNA (ssDNA) generated during DNA replication, recombination, and repair is rapidly bound and protected by the major single-stranded DNA-binding protein replication protein A (RPA). RPA not only stabilizes ssDNA but also acts as a central platform that coordinates diverse DNA transactions. Exhaustion of RPA due to unregulated ssDNA production leads to replication fork breakage and replication catastrophe, underscoring its critical role in genome stability. However, the direct consequences of RPA limitation remain incompletely understood. Using Xenopus egg extracts, we show that excess ssDNA induces spontaneous priming, a reaction that is otherwise prevented in a physiological nuclear environment. We provide evidence that priming suppression is mediated by stoichiometric binding of RPA to ssDNA. Analysis of the ssDNA-binding proteome reveals that RPA promotes the association of ATR checkpoint factors, Polα–primase, and the RFWD3 ubiquitin ligase with ssDNA. In contrast, RPA depletion induces the recruitment of Rad51, Rad51 paralogs, and Fbh1, a DNA helicase that interacts with both RPA and Rad51 and promotes fork breakage under replication stress. Collectively, our findings suggest that RPA contributes to genome stability by protecting ssDNA from inappropriate DNA synthesis and unscheduled recruitment of recombination and fork-processing factors.
2025
2.
Daichi Nishiguchi, Kensuke Tatsukawa, Tatsuro S. Takahashi
Preparation of Nucleoplasmic Extract and Its Application in DNA End Processing Book Chapter
In: Methods in Molecular Biology, pp. 201–223, Springer US, 2025, ISBN: 9781071647141.
Abstract | Links | タグ: Takahashi G
@inbook{Nishiguchi2025,
title = {Preparation of Nucleoplasmic Extract and Its Application in DNA End Processing},
author = {Daichi Nishiguchi and Kensuke Tatsukawa and Tatsuro S. Takahashi},
doi = {10.1007/978-1-0716-4714-1_14},
isbn = {9781071647141},
year = {2025},
date = {2025-08-21},
urldate = {2025-08-21},
booktitle = {Methods in Molecular Biology},
pages = {201--223},
publisher = {Springer US},
abstract = {The nucleoplasmic extract (NPE) of Xenopus laevis eggs contains a physiological concentration of nuclear proteins that recapitulate a functional nuclear environment. This system has been widely used to study key nuclear processes, including DNA replication, repair, transcription, and chromatin assembly. Its soluble nature, free from membrane fractions and insoluble structures, enables direct analysis of nuclear responses to specific DNA structures and lesions. Here, we describe an NPE preparation protocol with our modifications and its application in the processing of DNA double-strand breaks, a critical step in homology-directed repair.},
keywords = {Takahashi G},
pubstate = {published},
tppubtype = {inbook}
}
The nucleoplasmic extract (NPE) of Xenopus laevis eggs contains a physiological concentration of nuclear proteins that recapitulate a functional nuclear environment. This system has been widely used to study key nuclear processes, including DNA replication, repair, transcription, and chromatin assembly. Its soluble nature, free from membrane fractions and insoluble structures, enables direct analysis of nuclear responses to specific DNA structures and lesions. Here, we describe an NPE preparation protocol with our modifications and its application in the processing of DNA double-strand breaks, a critical step in homology-directed repair.
1.
Karin Shigenobu-Ueno, Reihi Sakamoto, Eiichiro Kanatsu, Yoshitaka Kawasoe, Tatsuro S Takahashi
In: The Journal of Biochemistry, 2025, ISSN: 1756-2651.
Abstract | Links | タグ: Takahashi G
@article{Shigenobu-Ueno2025,
title = {Replication across \textit{O}6-methylguanine activates futile cycling of DNA mismatch repair attempts assisted by the chromatin remodeling enzyme Smarcad1},
author = {Karin Shigenobu-Ueno and Reihi Sakamoto and Eiichiro Kanatsu and Yoshitaka Kawasoe and Tatsuro S Takahashi},
doi = {10.1093/jb/mvaf007},
issn = {1756-2651},
year = {2025},
date = {2025-01-30},
urldate = {2025-01-30},
journal = {The Journal of Biochemistry},
publisher = {Oxford University Press (OUP)},
abstract = {<jats:title>Abstract</jats:title>
<jats:p>SN1-type alkylating reagents generate O6-methylguanine (meG) lesions that activate the mismatch repair (MMR) response. Since post-replicative MMR specifically targets the nascent strand, meG on the template strand is refractory to rectification by MMR and, therefore, can induce non-productive MMR reactions. The cycling of futile MMR attempts is proposed to cause DNA double-strand breaks in the subsequent S phase, leading to ATR-checkpoint-mediated G2 arrest and apoptosis. However, the mechanistic details of futile MMR cycling, especially how this reaction is maintained in chromatin, remain unclear. Using replication-competent Xenopus egg extracts, we herein establish an in vitro system that recapitulates futile MMR cycling in the chromatin context. The meG–T mispair, but not the meG–C pair, is efficiently targeted by MMR in our system. MMR attempts on the meG-strand result in the meG-to-A correction, while those on the T-strand induce iterative cycles of strand excision and resynthesis. Likewise, replication across meG generates persistent single-strand breaks on the daughter DNA containing meG. Moreover, the depletion of Smarcad1, a chromatin remodeler previously reported to facilitate MMR, impairs the retention of single-strand breaks. Our study thus provides experimental evidence that chromatin replication across meG induces futile MMR cycling that is assisted by Smarcad1.</jats:p>},
keywords = {Takahashi G},
pubstate = {published},
tppubtype = {article}
}
<jats:title>Abstract</jats:title>
<jats:p>SN1-type alkylating reagents generate O6-methylguanine (meG) lesions that activate the mismatch repair (MMR) response. Since post-replicative MMR specifically targets the nascent strand, meG on the template strand is refractory to rectification by MMR and, therefore, can induce non-productive MMR reactions. The cycling of futile MMR attempts is proposed to cause DNA double-strand breaks in the subsequent S phase, leading to ATR-checkpoint-mediated G2 arrest and apoptosis. However, the mechanistic details of futile MMR cycling, especially how this reaction is maintained in chromatin, remain unclear. Using replication-competent Xenopus egg extracts, we herein establish an in vitro system that recapitulates futile MMR cycling in the chromatin context. The meG–T mispair, but not the meG–C pair, is efficiently targeted by MMR in our system. MMR attempts on the meG-strand result in the meG-to-A correction, while those on the T-strand induce iterative cycles of strand excision and resynthesis. Likewise, replication across meG generates persistent single-strand breaks on the daughter DNA containing meG. Moreover, the depletion of Smarcad1, a chromatin remodeler previously reported to facilitate MMR, impairs the retention of single-strand breaks. Our study thus provides experimental evidence that chromatin replication across meG induces futile MMR cycling that is assisted by Smarcad1.</jats:p>
<jats:p>SN1-type alkylating reagents generate O6-methylguanine (meG) lesions that activate the mismatch repair (MMR) response. Since post-replicative MMR specifically targets the nascent strand, meG on the template strand is refractory to rectification by MMR and, therefore, can induce non-productive MMR reactions. The cycling of futile MMR attempts is proposed to cause DNA double-strand breaks in the subsequent S phase, leading to ATR-checkpoint-mediated G2 arrest and apoptosis. However, the mechanistic details of futile MMR cycling, especially how this reaction is maintained in chromatin, remain unclear. Using replication-competent Xenopus egg extracts, we herein establish an in vitro system that recapitulates futile MMR cycling in the chromatin context. The meG–T mispair, but not the meG–C pair, is efficiently targeted by MMR in our system. MMR attempts on the meG-strand result in the meG-to-A correction, while those on the T-strand induce iterative cycles of strand excision and resynthesis. Likewise, replication across meG generates persistent single-strand breaks on the daughter DNA containing meG. Moreover, the depletion of Smarcad1, a chromatin remodeler previously reported to facilitate MMR, impairs the retention of single-strand breaks. Our study thus provides experimental evidence that chromatin replication across meG induces futile MMR cycling that is assisted by Smarcad1.</jats:p>
