Molecular Dissection of Human DNA Excision Repair Genes

1.利用酵母双杂交系统，我们克隆了一个新的XPA结合蛋白基因，命名为XAB 2。XAB 2与XPA的结合通过使用GST-XPA和体外翻译的XAB 2的体外下拉测定来证实。此外，XAB 2通过抗XPA抗体与来自HeLa细胞提取物的XPA免疫共沉淀，并且XPA通过抗XAB 2抗体与XAB 2免疫共沉淀，证实了XPA和XAB 2在细胞中的缔合。由于CSA和CSB在基础转录或转录偶联修复方面存在缺陷，因此XAB 2可能参与了CSA和CSB的修复机制（Tanaka，Wood和Hoeijmakers）。2.我们建立了XPA或CSB缺陷小鼠，但它们本身没有表现出任何明显的生理异常或病理改变，尽管它们缺乏核苷酸切除然而，XPA/CSB双基因敲除小鼠体积小，出生后20天内死亡。3.我们发现XPC与酵母RAD 23的人类同源物HHR 23 B相关，并且HHR 23 B及其同源物HHR 23 A是核苷酸切除修复的基本过程所必需的（Hanaoka，Wood和Hoeijmakers）。建立该基因缺陷小鼠的基因靶向实验正在进行中（Yasui和Hoeijmakers）。

1.Using yeast two hybrid system, we cloned a gene designated XAB2 which encodes new XPA binding protein. The binding of XAB2 to XPA was confirmed by in vitro pull-down assay using GST-XPA and in vitro translated XAB2. Moreover, the XAB2 was co-immunoprecipitated with XPA from HeLa cell extract by anti-XPA antibody, and the XPA was co-immunoprecipitated with XAB2 by anti-XAB2 antibody, confirming the association of XPA and XAB2 in the cells. We also found that the XAB2 associates with CSA and CSB.Since CSA and CSB have a defect in basal transcription or transcription coupled repair, it was strongly suggested that the XAB2 is involved in this mechanism (Tanaka, Wood and Hoeijmakers).2.We established the XPA-or CSB-deficient mice, but they did not show any significant physical abnormalities or pathological alterations by themselves although they are dificient in nucleotide excision repair and sensitive to UV.However, XPA/CSB double knockout mice were small and died in 20 days after birth. This result suggests that XPA and CSB belong to different epistasis groups (Tanaka and Hoeijmakers).3.We found that XPC associates with HHR23B,a human homolog of yeast RAD23 and that the HHR23B and its homolog HHR23A are indispensable to basic process of uncleotide excision repair (Hanaoka, Wood and Hoeijmakers).4.Human and mouse homologs of photoreactivating enzyme gene have been cloned. Gene targeting experiment to establish the mice which are deficient in this gene is in progress (Yasui and Hoeijmakers).