Background

Cells are continuously exposed to different sources of DNA damage. The nucleotide excision repair (NER) pathway comprises two sub pathways, transcription-coupled repair (TCR) and global genome repair (GGR), which remove helix-distorting lesions including UV-induced photoproducts from genomic DNA. While TCR preferentially removes DNA lesions from the transcribed strand of active genes (van den Heuvel et al., 2021), GGR removes lesions throughout the genome and is the dominant contributor to overall cellular NER activity (Marteijn et al., 2014). Both pathways funnel into a common molecular mechanism involving the same set of core NER proteins that mediate double incision by endonucleases ERCC1-XPF and XPG, resulting in the removal of a 30-nucleotide stretch of single-stranded DNA containing the lesion (van Toorn et al., 2022). After dual incision, the resulting gap is filled by DNA polymerases and sealed by DNA ligases (Ogi et al., 2010). DNA repair–associated DNA synthesis is distinct from synthesis during the S-phase to duplicate the genome, and is therefore referred to as unscheduled DNA synthesis (UDS) (Lehmann and Stevens, 1980). Irradiation with UV-C light generates two main types of genomic lesions: 6–4 pyrimidine–pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). The 6-4PPs are repaired considerably faster (within ∼5 h) than the CPDs, which are still present 24 h after UV irradiation (van Hoffen et al., 1995; Luijsterburg et al., 2010; Verbruggen et al., 2014). UDS after UV irradiation can be directly measured via nucleotide incorporation in non-dividing cells and mainly reflects GGR activity, which is responsible for approximately 90% of the overall cellular NER activity (Limsirichaikul et al., 2009). Although radioactive nucleotide analogues were initially used to measure UDS (Cleaver, 1968; Friedberg, 2004), the use of alkyne-linked thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) is more common nowadays (Limsirichaikul et al., 2009). The incorporated EdU can be visualized by its coupling to a fluorescent azide via Click-iT chemistry (Salic and Mitchison, 2008). Measuring UDS can offer insight in the potential involvement of GGR regulators or assess the functionality of mutated NER proteins, as recently reported for chromatin remodeler ALC1 or the ERCC1^R156W variant found in patients with liver and kidney dysfunction, respectively (Blessing et al., 2022;Apelt et al., 2021). In this protocol, we describe a straightforward and accessible method for measuring UDS at sites of local UV irradiation. In brief, cells are exposed to local UV-C irradiation through a micropore filter, causing DNA damage induction and NER-dependent UDS in sub-nuclear regions. The UDS signal at sites of local UV damage can be captured by pulse-labeling with EdU within the first few hours after UV irradiation, followed by fixation and visualization of the incorporated EdU by fluorescent Click-iT chemistry. NER-dependent DNA synthesis mainly reflects rapid 6-4PP repair within the first hours after UV irradiation, during which CPD repair is negligible (van Hoffen et al., 1995; Luijsterburg et al., 2010; Verbruggen et al., 2014). The simultaneous immunodetection of CPD photolesions, which are largely unrepaired within the first hours after UV irradiation, can be used to define sites of local damage. The use of local UV irradiation enables a per-cell correction for unspecific EdU incorporation and accurate exclusion of replicating cells with scheduled EdU incorporation throughout the nucleus. Moreover, we provide a custom-built ImageJ plug-in to quantify UDS signals and apply a correction for potential differences in DNA damage induction between experimental conditions based on the CPD signal. We show that local UDS at sites of UV-induced DNA damage is robustly detected in RPE1-hTERT human cells, while the UDS signal is nearly abolished in an isogenic knockout cell-line for essential NER geneXPG.