Mutations in XPA that prevent association with ERCC1 are defective in nucleotide excision repair.
The human repair proteins XPA and ERCC1 have been shown to be absolutely required for the incision step of nucleotide excision repair, and recently we identified an interaction between these two proteins both in vivo and in vitro (L. Li, S. J. Elledge, C. A. Peterson, E. S. Bales, and R. J. Legerski, Proc. Natl. Acad. Sci. USA 91:5012-5016, 1994). In this report, we demonstrate the functional relevance of this interaction. The ERCC1-binding domain on XPA was previously mapped to a region containing two highly conserved XPA sequences, Gly-72 to Phe-75 and Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these motifs and create two XPA mutants referred to as delta G and delta E. In vitro, the binding of ERCC1 to delta E was reduced by approximately 70%, and binding to delta G was undetectable; furthermore, both mutants failed to complement XPA cell extracts in an in vitro DNA repair synthesis assay. In vivo, the delta E mutant exhibited an intermediate level of complementation of XPA cells and the delta G mutant exhibited little or no complementation. In addition, the delta G mutant inhibited repair synthesis in wild-type cell extracts, indicating that it is a dominant negative mutant. The delta E and delta G mutations, however, did not affect preferential binding of XPA to damaged DNA. These results suggest that the association between XPA and ERCC1 is a required step in the nucleotide excision repair pathway and that the probable role of the interaction is to recruit the ERCC1 incision complex to the damage site. Finally, the affinity of the XPA-ERCC1 complex was found to increase as a function of salt concentration, indicating a hydrophobic interaction; the half-life of the complex was determined to be approximately 90 min.
Li
L
L
Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Peterson
C A
CA
Lu
X
X
Legerski
R J
RJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004252:DNA Mutational Analysis; D004260:DNA Repair / Q000235:genetics* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases; D005799:Genes, Dominant; D005816:Genetic Complementation Test; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011506:Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C071447:ERCC1 protein, human; D004720:Endonucleases