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 nucleotide is necessary to hand over the damaged site to the next GGR player. Surprisingly, this mutagenesis analysis also reveals that the rapid intranuclear trafficking of XPC protein depends on constitutive interactions with native DNA, implying that the search for base damage takes place in living cells by a facilitated diffusion process. 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 nucleotide is necessary to hand over the damaged site to the next GGR player. Surprisingly, this mutagenesis analysis also reveals that the rapid intranuclear trafficking of XPC protein depends on constitutive interactions with native DNA, implying that the search for base damage takes place in living cells by a facilitated diffusion process.

Overview of xeroderma pigmentosum proteins architecture, mutations and post-translational modifications

“…Remarkably, the XPC (Phe797Ala) and XPC (Phe799Ala) mutants exhibited a decreased ability to recruit the TFIIH complex [17].…”

“…These four mutants also exhibited decreased levels of accumulation at UV-damaged sites compared with wild-type (WT) XPC. FRAP measurements reveals that these mutations restricted XPC nuclear mobility and prevented the formation of stable recognition complexes [17].…”

“…Site-directed mutagenesis of four conserved residues (Phe799Ala, Phe797Ala, Phe756Ala and Asp754Ala) in human XPC was used to evaluate DNA binding and DNA damage recognition [17] (Table 2). Nearly all of the heterologously expressed XPC mutants exhibited a reduced ability to bind undamaged DNA, with the exception of XPC (Phe799Ala), which retained DNA-binding properties [17].…”

“…Nearly all of the heterologously expressed XPC mutants exhibited a reduced ability to bind undamaged DNA, with the exception of XPC (Phe799Ala), which retained DNA-binding properties [17]. These four mutants also exhibited decreased levels of accumulation at UV-damaged sites compared with wild-type (WT) XPC.…”

“…XPC Asp754Ala, Phe756Ala, Phe797Ala and Phe799Ala: DNA-binding defect, unable to bind single-stranded oligonucleotides in undamaged DNA (except Phe799Ala); these mutations result in low accumulation of XPC at UV-damaged sites; Phe797Ala and Phe799Ala result in the inability of XPC to recruit the TFIIH complex[17]; Trp690Ser, Trp690Ala, Trp531Ala and Trp542Ala: DNA-binding defect[18,106]; Phe733Ala: nearly complete loss of XPC binding to DNA[18]; XPC 427-940 and XPC 607-940 : loss of XPC binding to DNA[18]; XPC 1-495 and XPC 1-718 : loss of XPC recognition of DNA damage[18];…”