Sabatella, Mariangela et al. (2015) International Worm Meeting "Analysis of Nucleotide Excision Repair in vivo."

Sabatella, Mariangela, Vermeulen, Wim, Lans, Hannes, Thijssen, Karen

[International Worm Meeting, 2015]

Nucleotide Excision Repair (NER) is a major genome maintenance mechanism responsible for removing DNA-distorting lesions, in particular those formed by UV light. NER is initiated by two distinct damage detection mechanisms: Global Genome NER (GG-NER), that detects damage anywhere in the genome, and Transcription Coupled NER (TC-NER), that detects damage in the transcribed strand during transcription. Both mechanisms converge in a common pathway that verifies the presence of damage, excises it and fills the gap. In humans, NER deficiency affects tissues in various manners and is associated with several clinical disorders that are characterized by cancer predisposition, developmental defects and accelerated aging. Although, the molecular mechanism of NER is known in detail, mainly from in vitro and cell culture experiments, it is not entirely clear how defects in one pathway lead to these various and tissue-specific symptoms.We use C. elegans to better understand how NER operates and is regulated in vivo, in different tissues and stages of development. Previously we have shown that GG-NER mainly acts in germ cells while TC-NER mainly acts in somatic cells to protect against UV-induced DNA damage. Here, we focus on the ERCC-1/XPF-1 complex, a structure-specific endonuclease involved in damage excision during NER but which also acts in the repair of interstrand crosslinks and double strand breaks. To determine how this endonuclease responds to DNA damage and to identify which mechanisms regulate this, we expressed fluorescently tagged XPF-1 in different tissues and in diverse NER genetics backgrounds. By imaging living and fixed animals, we analyze the in vivo kinetics of the repair reaction in response to different kinds of DNA damage. Preliminary results show that XPF-1 quickly re-localizes in response to DNA damage in different tissues. By performing genetic analysis and proteomic screening we hope to uncover the regulatory mechanisms that underlie cell type specific responses of NER.

Sabatella, Mariangela et al. (2021) International Worm Meeting "Tissue-specific DNA repair activity of ERCC-1/XPF-1"

Thijssen, Karen L., Lans, Hannes, Davo-Martinez, Carlota, Sabatella, Mariangela, Vermeulen, Wim

[International Worm Meeting, 2021]

Hereditary DNA repair defects affect tissues differently, suggesting that in vivo cells respond differently to DNA damage. However, knowledge of the DNA damage response is largely based on in vitro and cell culture studies. A prime example of a DNA repair defect leading to pleiotropic and tissue-specific symptoms, including neurodegeneration, developmental defects, cancer, bone marrow failure and accelerated aging, is deficiency of the ERCC1/XPF complex. ERCC1/XPF is a structure specific endonuclease that is involved in several DNA repair pathways and has a critical role in nucleotide excision repair (NER). This major DDR pathway is responsible for removing bulky DNA lesions, including those formed by UV light. Here, we use in vivo imaging of ERCC-1/XPF-1 in C. elegans to demonstrate tissue-specific NER activity. In oocytes, XPF-1 functions as part of global genome NER to ensure extremely rapid removal of DNA-helix distorting lesions throughout the genome. In contrast, in post-mitotic neurons and muscles, XPF-1 participates in NER of transcribed genes only. Strikingly, muscle cells appear more resistant to the effects of DNA damage than neurons. These results suggest a tissue-specific organization of the DNA damage response and may help to better understand pleiotropic and tissue-specific consequences of accumulating DNA damage.

Thijssen, Karen et al. (2021) International Worm Meeting "C. elegans TFIIH subunit GTF-2H5/TTDA is a non-essential transcription factor indispensable for DNA repair"

Lans, Hannes, Vermeulen, Wim, Sabatella, Mariangela, Davo-Martinez, Carlota, Thijssen, Karen, Woude, van der, Melanie

[International Worm Meeting, 2021]

The 10-subunit transcription factor TFIIH is vital to both transcription initiation and nucleotide excision repair. In transcription initiation, TFIIH facilitates promoter escape and RNA synthesis by RNA polymerase II. In nucleotide excision repair, TFIIH facilitates DNA damage verification and subsequent excision by endonucleases ERCC1/XPF and XPG. Hereditary mutations in TFIIH subunits cause different diseases, including the cancer prone xeroderma pigmentosum and the progeroid Cockayne syndrome. Mutations in the smallest subunit of TFIIH, TTDA/GTF2H5, cause xeroderma pigmentosum combined with the rare developmental disorder trichothiodystrophy. Trichothiodystrophy is thought to be brought about by gene expression defects, but to which extent TTDA/GTF2H5 is necessary for transcription in vivo is unclear. Trichothiodystrophy patients express a partially functional TTDA/GTF2H5 protein whereas mice with complete TTDA/GTF2H5 loss are not viable. Therefore, TTDA/GTF2H5 has been considered to be essential to multicellular life. We investigated the function of C. elegans TFIIH and its GTF-2H5 subunit in transcription and DNA repair. We show that in contrast to full depletion of other TFIIH subunits, complete loss of GTF-2H5 is compatible with C. elegans viability and growth. However, GTF-2H5 is indispensable for nucleotide excision repair, in which it promotes recruitment of the TFIIH complex to DNA damage. Also, GTF-2H5 promotes the stability of TFIIH in multiple tissues. Because of this, GTF-2H5 loss causes embryonic lethality when transcription is challenged. These results support the idea that TTDA/GTF2H5 mutations cause transcription impairment that underlies trichothiodystrophy and establish C. elegans as potential model for studying the pathogenesis of this disease.

van der Woude M et al. (2021) STAR Protoc "C.elegans survival assays to discern global and transcription-coupled nucleotide excision repair."

Lans H, van der Woude M

[STAR Protoc, 2021]

Global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) protect cells against a variety of helix-distorting DNA lesions. In <i>C.elegans</i>, GG-NER primarily acts in proliferative germ cells and embryos, while TC-NER acts in post-mitotic somatic cells to maintain transcription. We leverage this difference to distinguish whether proteins function in GG-NER and/or TC-NER by straightforward UV survival assays. Here, we detail a protocol for these assays, using GG-NER factor <i>xpc-1</i> and TC-NER factor <i>csb-1</i> as examples. For complete details on the use and execution of this protocol, please refer to Sabatella etal. (2021).