Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA.
The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.
Camenisch
Ulrike
U
Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Winterthurerstrasse 260, 8057 Zürich, Switzerland.
Dip
Ramiro
R
Vitanescu
Mirela
M
Naegeli
Hanspeter
H
D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D016297:Mutagenesis, Site-Directed; D055672:Static Electricity; D051760:Xeroderma Pigmentosum Group A Protein / Q000235:genetics / Q000378:metabolism*
C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA