Anomalous DNA unwinding dynamics in mismatched DNA uncovered with laser temperature-jump spectroscopy and implicated in DNA damage sensing

Altered DNA dynamics at lesion sites are implicated in how DNA-repair proteins sense damage amid genomic DNA. We examined DNA dynamics in the context of damage recognition by NER protein XPC (yeast ortholog Rad4). Fluorescence lifetime studies on DNA with cytosine-analog FRET pair on either side of 3-bp mismatches – recognized by Rad4 as specific substrates in vitro – unveiled major deviations from B-DNA for high-specificity Rad4 substrates, even in the absence of Rad4.¹ With laser T-jump, we revealed the unwinding dynamics of these DNA. Specific, mismatched DNA showed large-amplitude kinetics over multiple timescales, including “missing amplitudes” outside our T-jump time-window – a fast (<20 μs) phase with 70-80% amplitude and an additional slower (>100 ms) phase appearing above ∼35 °C, which disappeared with Rad4 bound. We suggest that the <20-μs DNA fluctuations engage the protein at a “faulty” site and that the >100-ms kinetics reflect a propensity for specific DNA to adopt severely distorted conformations preferred by Rad4, albeit with high free energy barriers. Rad4, once engaged, lowers the barrier for full DNA distortions to form the recognition complex. These studies provide compelling evidence for unusual DNA dynamics at damaged sites that Rad4 can sense.