研究実績
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>
