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 temperaturejump spectroscopy. Besides identifying the~10-ms step as the ratelimiting 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.DNA damage recognition | time-resolved fluorescence spectroscopy | temperature-jump perturbation | DNA unwinding dynamics | xeroderma pigmentosum E ssential genetic mechanisms, such as replication, transcription, recombination, and repair, all require recognition of specific DNA sequences or structures by specialized proteins. How DNA-binding proteins search for and identify their target sites embedded in a vast excess of nontarget sites, especially if using only thermal energy, is a fundamental question in biology. Several lines of evidence indicate that proteins use some combination of 3D diffusion in the bulk solution and 1D diffusion while nonspecifically bound to DNA, and use this "facilitated diffusion" as a means to search efficiently in genomic DNA for their targets (1-7). Direct observations of proteins diffusing on nonspecific DNA have revealed residence times per base pair site ranging from 50 ns to 300 μs (7-13). On the other end, highresolution structures and thermodynamic studies on a wide range of specific protein-DNA complexes have revealed significant distortions in otherwise B-form DNA duplex structures and concerted rearrangements in the bound protein to accommodate the deformed DNA; this "induced-fit" mechanism has emerged as a general pr...