DNA damage repair starts with the recognition of damaged sites from 
predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental 
sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision 
repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) 
showed that Rad4 "opens" up damaged DNA by inserting a β-hairpin into the duplex 
and flipping out two damage-containing nucleotide pairs. However, this DNA 
lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond 
residence times per base pair site reported for various DNA-binding proteins 
during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to 
recognize lesions during diffusional search, we examine conformational dynamics 
along the lesion recognition trajectory using temperature-jump spectroscopy. 
Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards 
opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we 
assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The 
β-hairpin is not required to unwind or to overcome the bottleneck but is 
essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions 
in a step-wise "twist-open" mechanism, in which preliminary twisting represents 
Rad4 interconverting between search and interrogation modes. Through such 
conformational switches compatible with rapid diffusion on DNA, Rad4 may stall 
preferentially at a lesion site, offering time to open DNA. This study 
represents the first direct observation, to our knowledge, of dynamical DNA 
distortions during search/interrogation beyond base pair breathing. 
Submillisecond interrogation with preferential stalling at cognate sites may be 
common to various DNA-binding proteins.