Dissection of the xeroderma pigmentosum group C protein function by site-directed mutagenesis

Abstract Xeroderma pigmentosum group C (XPC) protein is a sensor of helix-distorting DNA lesions, the function of which is to trigger the global genome repair (GGR) pathway. Previous studies demonstrated that XPC protein operates by detecting the single-stranded character of non–hydrogen-bonded bases opposing lesion sites. This mode of action is supported by structural analyses of the yeast Rad4 homologue that identified critical side chains making close contacts with a pair of extrahelical nucleotides. Here, alanine substitutions of the respective conserved residues (N754, F756, F797, F799) in human XPC were tested for DNA-binding activity, accumulation in tracks and foci of DNA lesions, nuclear protein mobility, and the induction of downstream GGR reactions. This study discloses a dynamic interplay between XPC protein and DNA, whereby the association with one displaced nucleotide in the undamaged strand mediates the initial encounter with lesion sites. The additional flipping-out of an adjacent nucleoti...