Minimum Energy Pathway for Lesion Recognition and DNA Binding by RAD4/XPC

The pyrimidine-pyrimidone (6-4) photoproduct (6-4PP) is a UV-induced DNA lesion implicated in skin disorders and cancers. The repair protein XPC/RAD4 detects this lesion and initiates nucleotide excision repair to safeguard genomic integrity. While the X-ray crystallographic structure of the 6-4PP containing DNA-RAD4/XPC complex reveals DNA distortion and extrusion of the lesion and partner bases, the precise mechanism by which RAD4/XPC initiates lesion repair remains unclear. To investigate this, we employed molecular dynamics simulations, umbrella sampling, and the nudged elastic band method to map the minimum energy path (MEP) from RAD4's initial encounter with damaged DNA to its bound state. Our results reveal that the initial interrogation phase involves partial opening of the DNA, marked by the partial extrusion of the lesion while its partner bases largely remain intrahelical, accompanied by significant DNA unwinding near the damage site. This partially opened state represents the rate-limiting distortion, with 5' base flipping as the bottleneck for downstream events leading to the bound state. Upon overcoming the bottleneck, the DNA adopts a final untwisted conformation, with the lesion flipping out first, followed by the complete sequential flipping of the 5' base and then the 3' partner bases, while full β-hairpin insertion ultimately stabilizes the final bound DNA complex. The energetics and structural intermediates along the MEP provide key insights into conformational changes that drive lesion recognition, extrusion, and stable binding, advancing our understanding of nucleotide excision repair.