Mapping the recognition pathway of cyclobutane pyrimidine dimer in DNA by Rad4/XPC

Abstract

Abstract UV radiation-induced DNA damages have adverse effects on genome integrity and cellular function. The most prevalent UV-induced DNA lesion is the cyclobutane pyrimidine dimer (CPD), which can cause skin disorders and cancers in humans. Rad4/XPC is a damage sensing protein that recognizes and repairs CPD lesions with high fidelity. However, the molecular mechanism of how Rad4/XPC interrogates CPD lesions remains elusive. Emerging viewpoints indicate that the association of Rad4/XPC with DNA, the insertion of a lesion-sensing β-hairpin of Rad4/XPC into the lesion site and the flipping of CPD’s partner bases (5′-dA and 3′-dA) are essential for damage recognition. Characterizing these slow events is challenging due to their infrequent occurrence on molecular time scales. Herein, we have used enhanced sampling and molecular dynamics simulations to investigate the mechanism and energetics of lesion recognition by Rad4/XPC, considering multiple plausible pathways between the crystal structure of the Rad4–DNA complex and nine intermediate states. Our results shed light on the most likely sequence of events, their potential coupling and energetics. Upon association, Rad4 and DNA form an encounter complex in which CPD and its partner bases remain in the duplex and the BHD3 β-hairpin is yet to be inserted into the lesion site. Subsequently, sequential base flipping occurs, with the flipping of the 5′-dA base preceding that of the 3′-dA base, followed by the insertion of the BHD3 β-hairpin into the lesion site. The results presented here have significant implications for understanding the molecular basis of UV-related skin disorders and cancers and for paving the way for novel therapeutic strategies.