DNA damage induces long range changes to duplex structure—a non-protein start to damage detection?

DNA-binding proteins must quickly locate specific sites on DNA to enable replication, repair, and transcription. While sequence-specific recognition is well understood, the physical basis of structure-specific recognition remains unclear, limiting our understanding of DNA damage repair. Proteins must distinguish damaged sites within largely undamaged DNA; however, studying this is challenging due to DNA’s dynamic nature. We hypothesized that DNA damage causes changes in DNA structure, signalling protein recruitment. Using confocal single-molecule FRET, we analysed five DNA duplexes containing modifications: ribonucleotide, 8-oxoguanine, an abasic site, a nick, and a gap, which are all involved in the base excision repair pathway. Each construct was measured with nine dye pairs in triplicate to capture changes in bending, twisting, and stretching. An automated analysis pipeline processed 162 measurements, enabling rigorous statistical comparisons. All modifications altered FRET efficiencies compared to undamaged DNA, including the subtlest change: a single oxygen difference (ribo- versus deoxyribonucleotide). Abasic sites, nicks, and gaps had the greatest effects. These findings provide direct evidence that DNA damage affects duplex structure and dynamics beyond the lesion site, suggesting DNA flexibility changes may act as an early signal for repair protein recruitment.