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

Abstract

Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins sense damage within genomic DNA. We examined DNA dynamics related to damage recognition by Rad4 (yeast ortholog of XPC), which recognizes diverse lesions from environmental mutagens and initiates nucleotide excision repair. Using laser temperature-jump (T-jump), we measured the dynamics of DNA containing 3 base-pair mismatches recognized specifically by Rad4 in vitro. The T-jump kinetics traces measured using a cytosine-analog FRET pair, together with rigorous comparison with equilibrium measurements, enabled conformational dynamics to be revealed beyond the T-jump observation window of [~]20 {micro}s - 50 ms. AT-rich nonspecific sites (matched or mismatched) exhibited dynamics primarily within the T-jump window, albeit with some amplitude in "missing" fast ( 50 ms) kinetics at elevated temperatures. We posit that the rapid ({micro}s-ms) fluctuations help stall a diffusing protein at AT-rich/damaged sites and that the >50-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.