Frederick G D, Amirkhan R H, Schultz R A, Friedberg E C
Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235.
Hum Mol Genet. 1994 Oct;3(10):1783-8. doi: 10.1093/hmg/3.10.1783.
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.
患有常染色体隐性疾病着色性干皮病（XP）的个体对阳光极度敏感，且在暴露部位易患皮肤癌。从XP患者身上培养的细胞对紫外线敏感，并且在受损DNA的核苷酸切除修复方面存在缺陷。这些细胞表型适合采用互补的实验策略，这种方法以前曾用于证明在引入XPD（ERCC2）基因后XP-D表型得到纠正。在本研究中，我们对XPD（ERCC2）基因的基因组结构进行了表征，发现它由23个外显子组成。这些数据有助于评估两个XP-D细胞系中等位基因的功能完整性。在GM436细胞系中，在XPD（ERCC2）cDNA的核苷酸位置1411处发现了一个C→G的颠换，预计这种变化会导致Leu461Val替代。XP67MA细胞系在第22外显子的核苷酸位置2176处的基因组DNA中发生了C→T的转换，在氨基酸726处引入了终止密码子TAG。预计后者会产生一个截短了34个氨基酸的蛋白质。尽管正常XPD cDNA的表达可以纠正XP-D细胞中的紫外线敏感表型，但携带这两种突变之一的cDNA构建体未能产生互补作用。这些结果证实了ERCC2在XP-D中的作用，并说明了利用细胞表型评估单核苷酸替代意义的能力。
