Structural and mutational analysis of the xeroderma pigmentosum group D (XPD) gene.
Individuals affected by the autosomal recessive disease xeroderma pigmentosum (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 DNA. 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 XPD (ERCC2) gene. In the present study, we have characterized the genomic organization of the XPD (ERCC2) gene and found it to be comprised of 23 exons. These data were helpful in evaluating the functional integrity of alleles in two XP-D cell lines. In cell line GM436 a C-->G transversion was found at nucleotide position 1411 in the XPD (ERCC2) cDNA, a change expected to result in a Leu461Val substitution. Cell line XP67MA 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 shown to correct the UV sensitivity phenotype in XP-D cells, cDNA constructs bearing either of the two mutations failed to yield complementation. These results confirm the role of ERCC2 in XP-D and illustrate the power of utilizing cellular phenotypes to evaluate the significance of single nucleotide substitutions.
Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235.
G D
GD
Frederick
R H
RH
Amirkhan
R A
RA
Schultz
E C
EC
Friedberg
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004265:DNA Helicases; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics; D004268:DNA-Binding Proteins; D005091:Exons; D005399:Fishes; D005808:Genes, Recessive; D006801:Humans; D007930:Leucine; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012878:Skin Neoplasms / Q000209:etiology; D014157:Transcription Factors; D014466:Ultraviolet Rays; D014633:Valine; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D051759:Xeroderma Pigmentosum Group D Protein
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011506:Proteins; D014157:Transcription Factors; D004247:DNA; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; C497292:ERCC2 protein, human; C497293:Ercc2 protein, mouse; D007930:Leucine; D014633:Valine