STUDY ON NOVEL REQULATORY MECHANISMA OF DNA REPAIR

为了研究DNA修复系统是如何调控的，我们分析了真核细胞中核苷酸切除修复(NER)和跨损伤合成(TLS)的分子机制，得到了以下结果：1)全球基因组NER的启动子XPC-HR23B蛋白复合体被发现与一个中心体蛋白中心有关。2)XPC-HR23B强烈识别DNA的特殊二级结构，涉及单链和双链连接。使用环状DNA底物的DNase I足迹分析表明，单个XPC-HR23B复合体以不对称的方式保护一个扭曲的位点。3)mHR23A KO小鼠没有表现出异常，而mHR23B KO小鼠表现出胚胎发育受损和高宫内或新生儿死亡率。存活的动物表现出多种异常，包括生长迟缓、面部畸形和雄性不育。4)在酵母双杂交筛选中发现XPC与胸腺嘧啶DNA糖基酶(TDG)相互作用。通过生化分析，XPC不仅在物理上与TDG相互作用，而且在功能上也与TDG相互作用。5)我们鉴定了萌芽酵母Rad4和人XPC的两个裂解酵母同源物，分别命名为Rfp4A和Rhp4B。6)人DNA聚合酶η(PolDNA聚合酶η)催化相对有效和准确的TLS通过5R-胸腺甘醇和5S-胸腺甘醇。7)在12 9个小鼠品系中，发现两个等位基因都存在零突变。在POLτ缺陷小鼠中，突变的总频率和频谱是正常的。

In order to investigate how DNA repair systems are regulated, we analysed molecular mechanisms of nucleotide excision repair (NER) and translesion synthesis (TLS) in eukaryotic cells, and obtained the following results.1) The XPC-HR23B protein complex, the initiator of global genome NER, was found to associate with a centrosome protein centrin 2.2) The XPC-HR23B strongly recognized specific secondary structures of DNA, involving a single-and double-strand junction. A DNase I footprint analysis, using a looped DNA substrate, revealed that a single XPC-HR23B complex protected a distorted site in an asymmetrical manner.3) While mHR23A KO mice showed no abnormalities, mHR23B KO mice showed impaired embryonic development and a high rate of intrauterine or neonatal death. Surviving animals display a variety of abnormalities, including retarded growth, facial dysmorphology, and male sterility.4) XPC was found to interact with thymine DNA glycosylase (TDG) in yeast two hybrid screening. By biochemical analyses, XPC interacted with TDG not only physically but also functionally.5) We identified two fission yeast homologs of budding yeast Rad4 and human XPC, designated Rfp4A and Rhp4B. Rhp4A was found to play roles in GGR and TCR, while Rhp4B acts as an accessory protein in GGR.6) Human DNA polymerase η (Pol η) catalyzed relatively efficient and accurate TLS past 5R-thymineglycol and 5S-thymineglycol.7) Both alleles of DNA polymerase τ (Pol τ) gene was found to have null mutation in 129-derived strains of mice. Overall frequency and spectrum of mutation were normal in Pol τ-deficient mice.