RT Journal Article
SR Electronic
T1 Recovery of protein synthesis to measure transcription-coupled DNA repair in living cells and tissues
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.01.24.525327
DO 10.1101/2023.01.24.525327
A1 van der Woude, Melanie
A1 Davó-Martínez, Carlota
A1 Thijssen, Karen L.
A1 Vermeulen, Wim
A1 Lans, Hannes
YR 2023
UL http://biorxiv.org/content/early/2023/01/24/2023.01.24.525327.abstract
AB 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.Competing Interest StatementThe authors have declared no competing interest.