Evidence for intrinsic DNA dynamics and deformability in damage sensing by the Rad4/XPC nucleotide excision repair complex

Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins pause and identify damage within genomic DNA. We examined DNA dynamics in the context of damage recognition by Rad4 (yeast ortholog of XPC), which recognizes diverse lesions from environmental mutagens and initiates nucleotide excision repair. Previous studies with a cytosine-analog FRET pair placed on either side of 3 base-pair (bp) mismatched sites – recognized specifically by Rad4 in vitro – unveiled severely deformed DNA even without Rad4 (Chakraborty et al. (2018) Nucleic Acid Res. 46: 1240-1255). Here, using laser T-jump, we revealed the timescales of these spontaneous deformations. 3-bp AT-rich nonspecific sites, whether matched or mismatched, exhibited conformational dynamics primarily within the T-jump observation window (∼20 µs – <100 ms), albeit with some amplitude in unresolved (<20 µs) kinetics. The amplitudes of the “missing” fast kinetics increased dramatically for mismatched specific sites, which were further distinguished by additional “missing” amplitude in slow (>100 ms) kinetics at elevated temperatures. We posit that the rapid (µs-ms) fluctuations help stall a diffusing protein at AT-rich/damaged sites and that the >100-ms kinetics reflect a propensity for specific DNA to adopt unwound/bent conformations that may resemble Rad4-bound structures. These studies provide compelling evidence for unusual DNA dynamics and deformability that likely govern how Rad4 senses DNA damage.