Biology subjects

Vermeulen, W.

Publications and source records attributed to Vermeulen, W..

8 recordsLinked to original sources

bioRxiv2026

Helicase-deficient TFIIH causes severe disease features via persistent DNA excision without damage removal

Nucleotide excision repair (NER) removes helix-distorting DNA lesions through the ten subunit TFIIH complex, whose XPB and XPD translocase/helicase activities unwind DNA to enable damage verification and subsequent endonucleolytic DNA incisions. While most XPD mutations cause xeroderma pigmentosum, specific helicase-deficient mutations cause severe Cockayne syndrome (CS) features, including progressive neurodegeneration, for which the basis remains unclear. Here we show that loss of XPD helicase activity traps TFIIH in a futile repair cycle in which DNA is incised at the wrong position, leading to repeated DNA excision and resynthesis without removal of the lesion. Using C. elegans, we find that this futile DNA excision cycle produces severe neuronal dysfunction in vivo that depends on transcription-coupled NER activity and is rescued by preventing recruitment of helicase deficient TFIIH. These findings demonstrate that NER incisions can occur without XPD mediated damage verification and that persistent futile DNA excision cycles cause severe disease features, indicating that persistent NER intermediates are more pathogenic than unrepaired DNA lesions.

genetics↗

bioRxiv2025

RNA polymerase II processing facilitates DNA repair and prevents DNA damage-induced neuronal and developmental failure

Hereditary transcription-coupled nucleotide excision repair (TC-NER) defects cause severe developmental and neurodegenerative features, as observed in Cockayne syndrome (CS), or mild cutaneous UV sensitivity, as observed in UV-sensitive syndrome. The mechanisms underlying the strikingly different clinical features of these syndromes are not fully understood. Using C. elegans, we demonstrate that TC-NER deficiency leads to DNA damage-induced motoneuronal and developmental failure, primarily caused by the lack of lesion removal due to persistent lesion-stalling of RNA polymerase II. If, in the absence of TC-NER, lesion-stalled RNA polymerase II is processed and removed, global genome NER acts as backup pathway to repair transcription-blocking lesions and prevents DNA damage-induced developmental failure. Our results furthermore show that processing of lesion-stalled RNA Polymerase II facilitates TC-NER and involves the activity of multiple E3 ubiquitin ligases. These findings reveal that persistently stalled RNA polymerase II, rather than TC-NER deficiency, is the major driver of severe disease features associated with TC-NER defects.

molecular biology↗

bioRxiv2024

Regulation of XPG-DNA damage binding dynamics by pre- and post-incision nucleotide excision repair factors and EXO1

The XPG endonuclease plays a crucial role in nucleotide excision repair (NER) and other genome maintenance pathways. Precise regulation of XPG recruitment and activity during DNA repair is essential to avoid erroneous DNA incisions and genomic instability. In this study, we employed live-cell imaging to investigate how XPG function is regulated during NER, focusing on its dynamic interactions with key factors involved in the pre- and post-incision steps. We found that TFIIH and XPA facilitate the recruitment and association of XPG with DNA damage, and that XPG localizes separately from TFIIH to UV-induced lesions. Furthermore, our results show that XPGs dissociation from DNA damage is triggered by its own incision activity as well as by that of XPF. Additionally, the exonuclease EXO1 promotes XPG dissociation, likely by processing incised DNA, even in the absence of XPG-mediated incision. Our findings help to better understand the regulatory mechanisms that control XPG activity during NER and provide important insights into the complex dynamics of the repair process.

genetics↗

bioRxiv2024

STK19 drives Transcription-Coupled Repair by stimulating repair complex stability, Pol II ubiquitylation and TFIIH recruitment

DNA damage forms a major obstacle for gene transcription by RNA polymerase II (Pol II). Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates transcription-blocking lesions (TBLs), thereby safeguarding accurate transcription, preserving correct cellular function and counteracting aging. TC-NER initiation involves the recognition of lesion-stalled Pol II by CSB, which recruits the CRL4CSA E3 ubiquitin ligase complex and UVSSA. TBL-induced ubiquitylation of Pol II at lysine 1268 of the RPB1 subunit by CRL4CSA serves as a critical TC-NER checkpoint, governing Pol II stability and initiating TBL excision by TFIIH recruitment. However, the precise regulatory mechanisms of the CRL4CSA E3 ligase activity and TFIIH recruitment remain elusive. Here, we reveal Inactive Serine/Threonine Kinase 19 (STK19) as a novel TC-NER factor, that is essential for correct TBL removal repair and subsequent transcription restart. Cryo-EM studies demonstrate that STK19 is an integral part of the Pol II-TC-NER complex, bridging CSA with UVSSA, RPB1 and downstream DNA. Live-cell imaging and interaction studies show that STK19 stimulates TC-NER complex stability and CRL4CSA activity, resulting in efficient Pol II ubiquitylation and correct UVSSA and TFIIH binding. These findings underscore the crucial role of STK19 as a core component of the TC-NER machinery and its key involvement in the cellular responses to DNA damage that interfere with transcription.

molecular biology↗

bioRxiv2023

Recovery of protein synthesis to measure transcription-coupled DNA repair in living cells and tissues

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that protects against the negative effects of transcription-blocking DNA lesions. Hereditary TC-NER deficiency causes pleiotropic and often severe neurodegenerative and progeroid symptoms. Multiple assays have been developed for the clinic and for research to measure TC-NER activity, which is hampered by the relatively low abundance of repair events taking place in transcribed DNA. Recovery of RNA Synthesis is widely used as indirect TC-NER assay based on the notion that lesion-blocked transcription only resumes after successful TC-NER. Here, we show that measuring novel synthesis of a protein that has been degraded prior to DNA damage induction is an equally effective but more versatile manner to indirectly monitor TC-NER. This Recovery of Protein Synthesis (RPS) assay is readily adaptable for use with different degradable proteins and readouts, including fluorescence imaging and immunoblot. Moreover, with the RPS assay TC-NER activity can be measured in real-time, in various living cells types and even in differentiated tissues of living organisms. As example, we show that TC-NER capacity declines in aging muscle tissue of C. elegans. Therefore, the RPS assay constitutes an important novel clinical and research tool to investigate transcription-coupled DNA repair.

genetics↗

bioRxiv2021

C. elegans TFIIH subunit GTF-2H5/TTDA is a non-essential transcription factor indispensable for DNA repair

The 10-subunit TFIIH complex is vital to both transcription initiation and nucleotide excision repair. Hereditary mutations in its smallest subunit, TTDA/GTF2H5, cause a photosensitive form of the rare developmental brittle hair disorder trichothiodystrophy (TTD). Some TTD features are thought to be caused by subtle transcription or gene expression defects. Strikingly, TTDA/GTF2H5 knockout mice are not viable, which makes it difficult to investigate how TTDA/GTF2H5 promotes transcription in vivo. Here, we show that deficiency of the C. elegans TTDA ortholog GTF-2H5 is, however, compatible with viability and growth, in contrast to depletion of other TFIIH subunits. We also show that GTF-2H5 promotes the stability of TFIIH in multiple tissues and is indispensable for nucleotide excision repair, in which it facilitates recruitment of the TFIIH complex to DNA damage. Strikingly, when transcription is challenged, gtf-2H5 embryos die due to the intrinsic TFIIH fragility in the absence of GTF-2H5. These results support the idea that TTDA/GTF2H5 mutations cause transcription impairment underlying trichothiodystrophy and establish C. elegans as potential model for studying the pathogenesis of this disease.

genetics↗

bioRxiv2021

Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA Polymerase II (Pol II), causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions (TBLs). However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR/cas9 screen, we identified elongation factor ELOF1 as an important new factor in the transcription stress response upon DNA damage. We show that ELOF1 has an evolutionary conserved role in Transcription-Coupled Nucleotide Excision Repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair TBLs and resume transcription. Additionally, ELOF1 modulates transcription to protect cells from transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage by two distinct mechanisms.

cell biology↗

bioRxiv2019

Cell-type specific concentration regulation of the basal transcription factor TFIIH

The basal transcription/repair factor TFIIH is a ten sub-unit complex essential for RNA polymerase II (RNAP2) transcription initiation and DNA repair. In both these processes TFIIH acts as a DNA helix opener (by the enzymatic activity of the XPB and XPD helicases), required for promoter escape of RNAP2 in transcription initiation, and to set the stage for strand incision within the Nucleotide Excision Repair (NER) pathway. We generated a knock-in mouse model that endogenously expresses a fluorescent version of XPB. Here we demonstrate, via confocal imaging of ex vivo tissues and cells derived from this mouse model, that TFIIH steady state levels are tightly regulated at the single cell level, thus keeping nuclear TFIIH concentrations remarkably constant in a cell type dependent manner. Moreover, we show that individual cellular TFIIH levels are proportional to the speed of mRNA production, hence to a cells transcriptional activity, which we can correlate to proliferation status. Importantly, cancer tissue presents a higher TFIIH than normal healthy tissues. Taken together, these results show that TFIIH cellular concentration might be used as a bona-fide marker of transcriptional activity and proliferation.\n\nSignificanceUsing a mouse model expressing a fluorescently version of TFIIH, we showed that TFIIH concentration is tightly controlled and that this concentration is proportional to the cellular transcriptional activity and proliferation capacity.

cell biology↗