STRUCTURAL AND MUTATIONAL ANALYSIS OF THE XERODERMA-PIGMENTOSUM GROUP-D (XPD) GENE

Individuals affected by the autosomal recessive disease xeroderma pigm entosum (XP) are acutely sensitive to sunlight and predisposed to skin cancer on exposed areas. Cells cultured from XP patients are both UV sensitive and defective in the nucleotide excision repair of damaged D NA. These cellular phenotypes are amenable to experimental strategies employing complementation, an approach previously used to demonstrate the correction of XP-D phenotypes following the introduction of the XP D (ERCC2) gene. In the present study, we have characterized the genomi c organization of the XPD (ERCC2) gene and found it to be comprised of 23 exons. These data were helpful in evaluating the functional integr ity of alleles in two XP-D cell lines. In cell line GM436 a C-->G tran sversion was found at nucleotide position 1411 in the XPD (ERCC2) cDNA , a change expected to result in a Leu461Val substitution. Cell line X P67MA carries a C-->T transition in genomic DNA at nucleotide position 2176 in exon 22, introducing the termination codon TAG at amino acid 726. The latter would be expected to produce a protein truncated by 34 amino acids. Although expression of the normal XPD cDNA could be show n to correct the UV sensitivity phenotype in XP-D cells, cDNA construc ts bearing either of the two mutations failed to yield complementation . These results confirm the role of ERCC2 in XP-D and illustrate the p ower of utilizing cellular phenotypes to evaluate the significance of single nucleotide substitutions.