Direct measurement of NER activity using sSTRIDE-NER

Nucleotide Excision Repair (NER) removes bulky DNA adducts and intrastrand crosslinks generated by ultraviolet light and chemotherapeutic agents such as cisplatin. NER capacity profoundly influences tumor response and resistance to DNA-damaging therapies. Despite its biological and clinical importance, direct and quantitative methods for measuring NER activity in situ are lacking. Current approaches provide indirect or bulk measurements that fail to capture the spatial and temporal dynamics of repair at the single-cell level. We developed sSTRIDE-NER, a new assay based on the STRIDE (SensiTive Recongition of Individual DNA Ends) platform, designed to visualize and measure single-strand breaks (SSBs) generated during active NER. The assay targets XPD, a core helicase of the TFIIH complex that localizes near incision sites where transient SSBs occur during the repair process. Incorporation of XPD detection into the STRIDE workflow enabled selective visualization of NER-associated DNA ends in individual nuclei. Validation experiments demonstrated high specificity: multiple negative technical controls yielded minimal background signal. Cisplatin was used as a positive control, as it induces bulky intrastrand crosslinks that robustly activate NER. Following cisplatin exposure, sSTRIDE-NER revealed a time-dependent increase in nuclear signal intensity, consistent with accumulation of NER-associated SSBs. sSTRIDE-NER provides the first single-cell, image-based method for direct measurement of NER activity through detection of SSBs proximal to XPD. This assay offers new opportunities to functionally profile NER capacity in cancer cell models and patient-derived samples. It can be applied to investigate mechanisms of resistance to platinum drugs, to evaluate DNA repair-targeting agents, and to support the development of functional biomarkers predictive of therapy response.