Language：English   Publishing type：Research paper (scientific journal)  

Stalling of elongating RNA polymerase II (RNAPII) at DNA lesions blocks transcription and triggers transcription-coupled repair (TCR). However, the mechanisms determining the fate of stalled RNAPII remain incompletely understood. Here, we develop a time-resolved assay to track RNAPII clearance and degradation at UV-induced lesions. We show that RNAPII ubiquitylation by CSB and the CRL4CSA ubiquitin ligase is essential, as loss of these proteins causes persistent RNAPII accumulation at damage sites. Downstream of CSB/CRL4CSA-mediated ubiquitylation, two distinct pathways mediate RNAPII removal. The primary rapid route relies on TFIIH, with its XPD helicase activity driving RNAPII dissociation after proper recruitment and positioning by ELOF1, UVSSA, and STK19. A secondary slow pathway is mediated by the ubiquitin-dependent segregase VCP, which compensates for impaired TFIIH function. While VCP contributes only minimally in TCR-proficient cells, inhibition of VCP in TFIIH-deficient contexts completely abrogates RNAPII clearance. Together, these findings establish a hierarchical program in which CSB/CRL4CSA-mediated ubiquitylation initiates RNAPII processing, TFIIH/XPD helicase activity provides the main clearance mechanism, and VCP-dependent extraction acts as a backup when TFIIH fails. This mechanistic framework explains how cells resolve DNA lesion-stalled RNAPII during normal and compromised TCR.

DOI： 10.1038/s41467-026-68413-4

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Language：English   Publishing type：Research paper (scientific journal)  

Few drugs are available for rare diseases due to economic disincentives. However, tailored medications for extremely-rare disorders (N-of-1) offer a ray of hope. Artificial antisense oligonucleotides (ASOs) are now best known for their use in spinal muscular atrophy (SMA). The success of nusinersen/Spinraza for SMA indicates ASO-therapies' potential for other rare conditions. We propose a strategy to develop N-of-1 ASOs for treating one form of trichothiodystrophy (TTD), a rare condition with multisystem abnormalities and reduced life expectancy, associated with instability and greatly reduced amounts of the DNA-repair/transcription factor TFIIH. The therapeutic target carry mutations in GTF2H5, encoding the TFIIH-p8 subunit. This approach was inspired by the diagnosis and molecular dissection of a xeroderma pigmentosum (XP) case with mutations in GTF2H4, encoding the TFIIH-p52 subunit. This is newly classified as a ninth XP complementation-group, XP-J, identified five decades after the discovery of the other XP complementation-groups. The p8-p52 interaction is required to support the TFIIH-complex formation, and the patient's p52 C-terminal truncation results in the complete absence of p8 in TFIIH. However, intriguingly, TFIIH remained stable in vivo, and the XP-J patient did not exhibit any TTD-features. The aim of our ASO-design is to induce a C-terminal truncation of p52 and we have successfully stabilised TFIIH in p8-deficient TTD-A patient cells.

DOI： 10.1172/jci195732

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The filamentous fungus Neurospora crassa is an organism that contains multiple nuclei in the asexual conidia and hyphae. Since the nuclei of dormant conidia are arrested at various points in the nuclear division cycle, it has been difficult to analyze drug sensitivity at the specific point of the cycle in N. crassa. In this study, we have stablished a useful method for analysis at different stages of the nuclear division cycle in N. crassa. This assay will be a reference for researchers to use the synchronized culture in other diverse analyses.

Open Access

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Ultraviolet (UV) light is a mutagen that causes DNA damage. Some UV-sensitive Neurospora crassa strains have been reported to exhibit a partial photoreactivation defect (PPD) phenotype, and the possible cause of this has been unknown for more than half a century. In this study, in the process of elucidating the possible causes of a PPD phenotype, we discovered that the XPF homologue MUS-38 is involved in repairing the UV-induced DNA interstrand crosslink (ICL) in N. crassa. Furthermore, the sensitivity of the Δmus-38 and Δmus-44 strains to ICL agents was significantly higher than that of other nucleotide excision repair (NER)-related gene knockout (KO) strains, indicating that the MUS-38/MUS-44 complex is involved in an NER-independent ICL repair mechanism. Based on reports concerning the mammalian homologues XPF and ERCC1 we obtained separation-of-function mutants defective only in NER in mus-38 and mus-44. Additionally, the photoreactivation ability of these mutants was significantly higher than that of the KO strains. These results indicate that the PPD phenotype is caused by a defect in the repair-ability of ICL induced by UV and that an NER-independent ICL repair by MUS-38 and MUS-44 confers resistance to UV in N. crassa.

DOI： 10.1016/j.fgb.2022.103752

PubMed

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Photoreactivation is a mechanism in which photolyase directly repairs either cyclobutane pyrimidine dimers (CPDs) or (6-4) photoproducts [(6-4) PPs] caused by ultraviolet (UV) light. In the filamentous fungus Neurospora crassa, some UV-sensitive mutants such as mus-44 have been reported to exhibit a partial photoreactivation defect (PPD) phenotype, but its mechanism has not been elucidated for a long time. In this study, the N. crassa CPD photolyase PHR was overexpressed in the Δmus-44 strain, but photoreactivation ability was not increased. Furthermore, Escherichia coli CPD photolyase or Arabidopsis thaliana (6-4) PP photolyase was also introduced into Δmus-44; however, the PPD phenotype was not complemented. These results suggested that the PPD phenotype in N. crassa is not caused by residual unrepaired pyrimidine dimers, which are the main type of DNA damage caused by UV irradiation. Finally, we revealed that Δmus-44, but not the Δmus-43 strain, which does not show the PPD phenotype, displayed higher sensitivity with increasing dose rate of UV. Moreover, Δmus-44 was also sensitive to an interstrand crosslinking agent. This indicates that the high dose of UV in our experimental condition induces DNA damage other than pyrimidine dimers, and that such damage is a likely cause of the PPD phenotype.

DOI： 10.1266/ggs.20-00022

Open Access

PubMed

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Authorship：Lead author,　Corresponding author   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Event date： 2025.5

Presentation type：Oral presentation (invited, special)  

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Event date： 2024.11

Language：English   Presentation type：Poster presentation  

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Event date： 2024.7

Presentation type：Poster presentation  

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Event date： 2024.6

Presentation type：Poster presentation  

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Event date： 2024.3

Language：English   Presentation type：Poster presentation  

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