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.

FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER

“…The amplified unscheduled DNA synthesis (UDS) assay was performed as described (36). Briefly, XP186LV, XPC-deficient cells, seeded on 24-mm coverslips 4 days prior to the experiment were transfected with siRNA 2 days later.…”

“…TC-NER-UDS signal was quantified by analyzing at least six fields for each condition Mean nuclear fluorescence signals were quantified using ImageJ software (Version 1.48) (37). Sample analysis was performed as described (36). The mean nuclear fluorescence signal ± standard error of the mean is shown.…”

A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair

“…TCR-specific unscheduled DNA synthesis. Detection of TCR-specific unscheduled DNA synthesis was performed essentially as previously described (Wienholz et al, 2017). Primary XP168LV (XP-C patient cells) were transfected with siRNAs and subsequently serum starved for at least 24 hrs in F10 medium (Lonza) supplemented with 0.5% FCS and antibiotics.…”

“…These XP-C cells were globally irradiated with UV-C light (8 J/m 2 ) and immediately pulse-labelled for 8 hours with the nucleotide analogue 5-ethynyl-deoxyuridine (EdU). TCR-specific UDS was visualized using Click-It chemistry combined with tyramide-based signal amplification (Wienholz et al, 2017). Robust incorporation of EdU could indeed be detected in UV-irradiated XP-C cells, which was virtually absent from non-irradiated controls cells ( Figure 3F-H).…”

Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair

“…TC-NER is often determined indirectly by quantifying the recovery of RNA synthesis (RRS) (35,36), or by using host cell reactivation assays (37). Alternatively, TC-NER can be measured in a direct manner by strand-specific repair assays (38), or by more recently developed single-cell assays, such as the modified COMET-FISH procedure (39), or the TC-NER specific UDS assay (40). Direct detection and quantification of UV-induced DNA damage and its removal in time can be accomplished using antibodies specifically recognizing CPD or 6-4PP lesions in combination with immunofluorescence or ELISA procedures (32).…”