Molecular Dissection of Human DNA Excision Repair Genes

人类 DNA 切除修复基因的分子解剖

基本信息

批准号：
07044265
负责人：
TANAKA Kiyoji
金额：
$ 5.95万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044265/
关键词：
DNA repair two hybrid system xeroderma pigmentosum gene targeting knockout mice Cockayne syndrome Yeast homolog ultraviolet photoreactivation DNA結合能 Znフィンガー RPA エンドヌクレアーゼ

项目摘要

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).

1.利用酵母二杂交系统，我们克隆了一个编码新的XPA结合蛋白的基因，命名为XAB2。 XAB2 与 XPA 的结合通过使用 GST-XPA 的体外 Pull-down 测定和体外翻译的 XAB2 得到证实。此外，通过抗XPA抗体将XAB2与来自HeLa细胞提取物的XPA进行免疫共沉淀，并通过抗XAB2抗体将XPA与XAB2进行免疫共沉淀，证实了XPA和XAB2在细胞中的关联。我们还发现 XAB2 与 CSA 和 CSB 相关。由于 CSA 和 CSB 在基础转录或转录偶联修复方面存在缺陷，因此强烈建议 XAB2 参与该机制（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身体异常或病理改变，尽管它们缺乏核苷酸切除修复并且对紫外线敏感。 XPA/CSB双基因敲除小鼠体型较小，出生后20天即死亡。这一结果表明XPA和CSB属于不同的上位基团(Tanaka和Hoeijmakers)。3.我们发现XPC与酵母RAD23的人类同源物HHR23B相关联，并且HHR23B及其同源物HHR23A对于不链核苷酸切除的基本过程是不可缺少的。修复（Hanaoka、Wood 和 Hoeijmakers）。4.人类和小鼠光活化酶基因的同源物已被克隆。用于建立缺乏该基因的小鼠的基因靶向实验正在进行中（Yasui 和 Hoeijmakers）。