Base-pair scale dynamics of a repair helicase on DNA lesions reveal varied damage-sensing mechanisms

Nucleotide excision repair (NER) is a cellular pathway that removes DNA lesions caused by ultraviolet light and various mutagens. A critical component of the NER machinery is XPD helicase, which unwinds the duplex around the damage, allowing its excision and repair. XPD has also been increasingly implicated in sensing and verifying DNA damage. However, the detailed mechanisms by which XPD responds to DNA damage have to-date remained unclear. Here, we use optical tweezers to perform real-time, high-precision measurements of single molecules of XPD as they encounter a variety of well-defined DNA modifications, including a cyclobutane pyrimidine dimer (CPD), a natural substrate for NER. The observed XPD dynamics reveal different behaviors depending on the damage type and the relative orientations of the DNA fork, damage, and helicase. Most notably, XPD displays an almost complete inability to unwind past a CPD on the translocated strand, and instead exhibits a short pause around the lesion-though not a stall-followed by retreat. Combining the base pair-scale XPD kinetics with structural analyses, we identify two regions of XPD sensitive to DNA modifications.