Gene diagnosis and establishment of mice model for xeroderma pigmentosum group A

我们克隆了A组着色性干皮病的致病基因(XPA基因)。XPA基因有一个大的开放阅读框，编码273个氨基酸的亲水性蛋白，带有C4型和H2C2型锌指基序。为了检测XPA蛋白的DNA修复功能，在大肠杆菌中表达了重组XPA蛋白，并通过凝胶迁移率改变和滤膜结合实验检测了XPA蛋白的DNA结合活性。XPA蛋白可与DNA结合，经紫外线、顺铂、乙酰氨基荧烯或三氧化二铯损伤的DNA结合的XPA蛋白多于未损伤的DNA，这些结果表明XPA蛋白参与了核苷酸切除修复过程中的损伤识别步骤。另一方面，我们在A组XP患者中发现了不同类型的XPA基因突变，并在此基础上进行了A组XP的基因诊断。然后，我们通过在小鼠ES细胞中靶向XPA基因，建立了XPA基因敲除小鼠。XPA纯合子小鼠的原代成纤维细胞对紫外线的杀伤敏感，对紫外线损伤的DNA的核苷酸切除修复有缺陷，而杂合子小鼠的成纤维细胞与野生型小鼠的成纤维细胞一样具有抵抗力。用7，12-二甲基苯并(A)菲(DMBA)治疗的XPA纯合子小鼠在治疗一周后出现严重的皮肤溃疡，此后出现肿瘤的频率很高。DMBA形成了巨大的DNA加合物。这些结果提供了一个直接的证据，证明XPA蛋白可以保护小鼠免受化学致癌物引起的持续的DNA损伤，并且小鼠是复制A组XP中肿瘤倾向的良好动物模型。

We have cloned a causative gene for group A xeroderma pigmentosum(XPA gene). The XPA cDNA has a single large open reading frame encoding hydrophilic protein of 273 amino acids with C4 type and H2C2 type zinc finger motifs. In order to examine DNA repair functions of the XPA protein, a recombinant XPA protein was produced in E.coli.DNA binding activities of the XPA protein were examined by gel mobility shift and filter binding assays. The XPA protein bound to DNA and more XPA protein bound to DNA damaged by ultraviolet light(UV), cisplatin, acetyl amino fluorene or osmium teraoxide than non damaged DNA.These results indicate that the XPA protein is involved in the damage-recognition step of nucleotide excision repair processes. On the other hand, we have found different types of mutations of the XPA gene in group A XP patients and executed gene diagnosis of group A XP on the basis of our results. Then, we have established XPA-knocked out mice by the targeting of the XPA gene in mouse ES cells. The primary fibroblasts derived from XPA-homozygous mice were hypersensitive to killing by UV and defective in nucleotide excision repair of DNA damaged by UV, while those from heterozygous mice were resistant as those from wild type mice. The XPA homozygous mice treated with 7, 12-Dimethylbenz(a)anthracene(DMBA), which forms bulky DNA adducts, developed severe skin ulcers one week after the treatment and tumors thereafter at high frequency. These results provide a direct evidence that the XPA protein protects mice from continued DNA damage elicited by a chemical carcinogen, and that the mice is a good animal model which reproduces tumor-proneness in group A XP.