Dissection of the xeroderma pigmentosum group C protein function by site-directed mutagenesis.
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.
Clement
Flurina C
FC
Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Winterthurerstrasse 260, Zürich, Switzerland.
Kaczmarek
Nina
N
Mathieu
Nadine
N
Tomas
Martin
M
Leitenstorfer
Alfred
A
Ferrando-May
Elisa
E
Naegeli
Hanspeter
H
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D051758:Transcription Factor TFIIH / Q000235:genetics / Q000378:metabolism; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D051758:Transcription Factor TFIIH; C086984:XPC protein, human