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

Abstract

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.