MUTATIONS IN XPA THAT PREVENT ASSOCIATION WITH ERCC1 ARE DEFECTIVE INNUCLEOTIDE EXCISION-REPAIR

The human repair proteins XPA acid ERCC1 have been shown to be absolut ely 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. Bale s, and R. J. Legerski, Proc. Natl. Acad. Sci. USA 91:5012-5016, 1991). In this report, we demonstrate the functional relevance of this inter action. The ERCC1-binding domain on XPA was previously mapped to a reg ion containing two highly conserved XPA sequences, Gly-72 to Phe-75 an d Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these moti fs and create two XPA mutants referred to as Delta G and Delta E. In v itro, the binding of ERCC1 to Delta E was reduced by approximately 70% , and binding to Delta G was undetectable; furthermore, both mutants f ailed to complement XPA cell extracts in an in vitro DNA repair synthe sis assay. In vivo, the Delta E mutant exhibited an intermediate level of complementation of XPA cells and the Delta G mutant exhibited litt le or no complementation. In addition, the Delta G mutant inhibited re pair synthesis in wild-type cell extracts, indicating that it is a dom inant 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 o f the interaction is to recruit the ERCC1 incision complex to the dama ged 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 approx imately 90 min.