Molecular Analysis of Human DNA Excision Repair Mechanism.

田中清二（Kiyoji Tanaka）、中津吉通（Yoshimichi西条）、Masafumi（Masafumi Saijo）等共同研究者访问了鹿特丹伊拉斯姆斯大学的Jan H. J. Hoeijmakers，讨论了着色性干皮病A组（XPA）和ERCC 1基因的DNA修复功能分析。田中和西条发现XPA蛋白是由273个氨基酸组成的锌金属蛋白，优先与紫外线或化学致癌物损伤的DNA结合，并鉴定了包含在XPA蛋白的截短衍生物MF 122中的XPA蛋白的DNA结合结构域，XPA蛋白由122个氨基酸组成，含有C4型锌指基序。这些结果表明，XPA蛋白的N端和C端区域可能具有重要的DNA修复功能，而不是DNA结合。我们推测它们可能是一种蛋白质的结构域，蛋白质相互作用来协调DNA切除修复过程，因此我们寻找与XPA蛋白质相互作用的蛋白质， ...更多信息 酵母双杂交系统。我们发现RPA ERCC 1和XPA蛋白之间的相互作用通过与Hoeijmakers的合作在体外测定中证实，与ERCC 1蛋白相互作用的XPA蛋白的结构域被分配到含有E-Tanaka还发现XPA和ERCC 1蛋白的相互作用导致XPA蛋白的损伤DNA结合活性增强。已经表明XPA蛋白可能在将切口复合物通过DNA结合位点装载到损伤DNA位点上中起作用。Tanaka利用ES细胞基因打靶技术建立了XPA基因缺失小鼠模型，该模型小鼠具有良好的生育能力，无明显的生理异常，但在DNA切除修复方面存在缺陷，当用DMBA处理或UVB照射后，Hoeijmaker已经培育出ERCC 1基因缺陷小鼠，这些小鼠在出生时发育不良，在断奶前死亡，这表明ERCC 1基因对于正常发育是必不可少的，因此XPA缺陷小鼠为研究体内紫外线或化学致癌和致突变提供了一个有用的模型。少

Kiyoji Tanaka, a head investigator, and Yoshimichi Nakatsu and Masafumi Saijo, collaborative investigators, have visited Jan H.J.Hoeijmakers at Erasmus University Rotterdam and discussed on the analyzes of DNA repair functions of xeroderma pigmentosum group A(XPA)and ERCC1 genes.Tanaka and Saijo found that the XPA protein is a zinc metalloprotein consisting of 273 amino acids which binds preferentially to UV-or chemical carcinogen-damaged DNA,and identified a DNA binding domain of the XPA protein which is contained within a truncated derivative of the XPA protein, MF122, consisting of 122 amino acids and containing a C4 type zinc finger motif.These results suggest that the N-terminal and C-terminal regions of the XPA protein may have an important DNA repair function other than DNA binding.We speculated that they might be domains for a protein-protein interaction to cordinate the DNA excision repair processes, and therefore we searched for proteins which interact with the XPA protein us … More ing yeast two hybrid system.We found that the RPA(replication protein A)and ERCC1 proteins interact with the XPA protein.The interaction between ERCC1 and XPA proteins was confirmed in in vitro assay by the collaboration with Hoeijmakers, and a domain of the XPA protein to interact with the ERCC1 protein was assigned to the N-terminal region containing E-cluster region.Tanaka also found that the interaction of the XPA and ERCC1 proteins caused an enhancement of the damaged DNA binding activity of the XPA protein.It has been suggested that the XPA protein may play a role in loading the incision complex onto a damaged DNA site by the interaction with ERCC1 protein.In addition, our results indicate that the interaction is also to increase the affinity of the repair protein complex for the damaged DNA.Tanaka has established the XPA deficient mice by gene targeting in ES cells.The homozygotes were fertile and showed no obvious physical abnormalities, but they were defective in the DNA excision repair.When they were treated with DMBA or irradiated with UVB,they developed papillomas and squamous cell carcinomas at higher frequency than wild type mice.Hoeijmaker has developed the ERCC1 deficient mice which were runted at birth and died before weaning, suggesting that the ERCC1 gene is indispensable for normal development.Thus the XPA-deficient mice provides a useful model to study UV-or chemical carcinogenesis and mutagenesis in vivo. Less