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Recovery of protein synthesis to assay DNA repair activity in transcribed genes in living cells and tissues

Nucleic Acids Res. 2023-10; 
Melanie van der Woude, Carlota Davó-Martínez, Karen L Thijssen, Wim Vermeulen, Hannes Lans
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Proteins, Expression, Isolation and Analysis … All reagents used in this study, with concentrations and identifiers, are listed in … The gene fragment was cloned into pUC57 (Genscript) and transfected together with Cas9 (IDT) and an … Get A Quote

摘要

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 deficiencies cause pleiotropic and often severe neurodegenerative and progeroid symptoms. While multiple assays have been developed to determine TC-NER activity for clinical and research purposes, monitoring TC-NER is hampered by the low frequency of repair events occurring 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 after its co... More

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