The cellular defense system known as global-genome nucleotide excision repair (GG-NER) safeguards genome stability by eliminating a plethora of structurally unrelated DNA adducts inflicted by chemical carcinogens, ultraviolet (UV) radiation or endogenous metabolic by-products. Xeroderma pigmentosum group C (XPC) protein provides the promiscuous damage sensor that initiates this versatile NER reaction through the sequential recruitment of DNA helicases and endonucleases, which in turn recognize and excise insulting base adducts. As a DNA damage sensor, XPC protein is very unique in that it (a) displays an extremely wide substrate range, (b) localizes DNA lesions by an entirely indirect readout strategy, (c) recruits not only NER factors but also multiple repair players, (d) interacts avidly with undamaged DNA, (e) also interrogates nucleosome-wrapped DNA irrespective of chromatin compaction and (f) additionally functions beyond repair as a co-activator of RNA polymerase II-mediated transcription. Many recent reports highlighted the complexity of a post-translational circuit that uses polypeptide modifiers to regulate the spatiotemporal activity of this multiuse sensor during the UV damage response in human skin. A newly emerging concept is that stringent regulation of the diverse XPC functions is needed to prioritize DNA repair while avoiding the futile processing of undamaged genes or silent genomic sequences.

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.

Histone octamers are the basic building blocks of chromatin and are platforms for diverse genetic mechanisms. We report a simple method for preparing recombinant histone octamers by overexpressing all four histones from a single polycistronic vector followed by standard chromatography under native conditions. This approach reduces the time needed for the octamer preparation to a single day and should be applicable to making a variety of unmodified and modified histone octamers.