Analysis of DNA repair functions of xeroderma pigmentosum gene

XPA基因是人类遗传性疾病A组着色性干皮病的致病基因，也是人类核苷酸切除修复(NER)的重要基因之一，编码273个氨基酸的蛋白质。我们发现XPA基因产物(XPA蛋白)优先与受损的DNA结合，提示其在NER的损伤识别步骤中发挥作用。受损的DNA结合活性定位于MF122蛋白(氨基酸残基98至219)包围的区域，MF122蛋白是一个离散折叠的功能迷你结构域，包含C4锌指基序。此外，XPA蛋白还具有与其他蛋白相互作用的结构域。利用酵母双杂交系统，我们发现ERCC1修复蛋白、复制蛋白A(RPA)等多种蛋白与XPA蛋白结合，XPA与ERCC1或RPA的相互作用增强了XPA蛋白的DNA结合活性，提示这种相互作用可能协调了NER的损伤识别步骤。我们正在制作缺乏这些基因的酵母突变体，以观察它们是否会显示出缺陷的NER。我们使用ES细胞中的基因打靶技术建立了XPA缺陷小鼠。它们在NER中存在缺陷，并且对紫外线或化学致癌物诱发的皮肤癌高度敏感。他们的大脑也更小。XPA基因缺陷小鼠可能为研究紫外光诱发皮肤癌变的多步骤过程和色素性干皮病患者神经功能障碍的分子基础提供有用的工具。

XPA gene is a causative gene for human genetic disease, xeroderma pigmentosum group A (XPA) and one of the important genes responsible for nucleotide excision repair (NER) in man and encodes a protein of 273 amino acids. We found that the XPA gene product (XPA protein) preferentially bound to the damaged DNA,suggesting its role in damage recognition step of NER.The damaged DNA binding activity is localized in the region encompassed by the MF122 protein (amino acid residues 98 to 219) which is a discretely folded, functional mini-domain, and contains C4 zinc-finger motif. Moreover, the XPA protein has domains to interact with other proteins. Using yeast two hybrid system, we found that the ERCC1 repair protein, replication protein A (RPA) and several other proteins bind to the XPA protein.The interaction between XPA and ERCC1 or RPA enhanced the damaged DNA binding activity of the XPA protein, suggesting that the interaction may coordinate the damage-recognition step of NER.Yeast homologs of new XPA-binding proteins have been cloned. We are making yeast mutants lacking these genes to see whether they will show a defective NER.We established XPA-deficient mice using gene targeting technology in ES cells. They are defective in NER and highly susceptible to ultraviolet light-or chemical carcinogen-induced skin carcinogeneis. They also had smaller brain. The XPA-deficient mice may provide a useful tool to study a multistep process of ultraviolet light-induced skin carcinogenesis and a molecular basis of the neurological disturbance in xeroderma pigmentosum patients.