"Flexible hinge" dynamics in mismatched DNA revealed by fluorescence lifetime and correlation spectroscopy

Enhanced fluctuations at DNA lesion sites are implicated in damage-sensing by DNA-repair proteins. We investigated the dynamics of DNA oligomers containing 3-bp mismatched sites specifically recognized in vitro by NER protein Rad4. A previous study mapped DNA conformational distributions using fluorescence lifetime (FLT) with cytosine-analog FRET pair sensitive to DNA unwinding (Chakraborty et al., 2018, NAR, 46, 1240). These studies revealed B-DNA conformations for low-specificity/nonspecific substrates but significant unwinding for high-specificity substrates, even in the absence of Rad4. The timescales of these unwinding fluctuations, however, remained elusive. Here, we labeled DNA with FRET dyes suitable for fluorescence correlation spectroscopy (FCS). FLT with these probes detected higher FRET in specific, mismatched DNA versus matched DNA, indicating bending deformations in the mismatched DNA. FCS uncovered ~100-300 µs dynamics on mismatched DNA with no dynamics detected for matched DNA, thus providing direct evidence of equilibrium unwinding/bending fluctuations in mismatched DNA on timescales that overlap with the <500 µs 1D stepping times of repair proteins diffusing on DNA. Such “flexible hinge” dynamics could arrest a diffusing protein to facilitate recognition.