title: Amplification of unscheduled DNA synthesis signal enables fluorescence-based single cell quantification of transcription-coupled nucleotide excision repair.
abstract: Nucleotide excision repair (NER) comprises two damage recognition pathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which remove a wide variety of helix-distorting lesions including UV-induced damage. During NER, a short stretch of single-stranded DNA containing damage is excised and the resulting gap is filled by DNA synthesis in a process called unscheduled DNA synthesis (UDS). UDS is measured by quantifying the incorporation of nucleotide analogues into repair patches to provide a measure of NER activity. However, this assay is unable to quantitatively determine TC-NER activity due to the low contribution of TC-NER to the overall NER activity. Therefore, we developed a user-friendly, fluorescence-based single-cell assay to measure TC-NER activity. We combined the UDS assay with tyramide-based signal amplification to greatly increase the UDS signal, thereby allowing UDS to be quantified at low UV doses, as well as DNA-repair synthesis of other excision-based repair mechanisms such as base excision repair and mismatch repair. Importantly, we demonstrated that the amplified UDS is sufficiently sensitive to quantify TC-NER-derived repair synthesis in GG-NER-deficient cells. This assay is important as a diagnostic tool for NER-related disorders and as a research tool for obtaining new insights into the mechanism and regulation of excision repair.
authors.affiliation: Department of Molecular Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.
authors.forename: Franziska
authors.initials: F
authors.lastname: Wienholz
authors.affiliation: Department of Molecular Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.
authors.forename: Wim
authors.initials: W
authors.lastname: Vermeulen
authors.affiliation: Department of Molecular Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.
authors.forename: Jurgen A
authors.initials: JA
authors.lastname: Marteijn
mesh_terms: D002460:Cell Line; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D005453:Fluorescence; D005821:Genetic Techniques; D006801:Humans; D014158:Transcription, Genetic; D014439:Tyramine / Q000737:chemistry
chemical_list: D014439:Tyramine