STUDY ON NOVEL REQULATORY MECHANISMA OF DNA REPAIR

DNA修复新调节机制的研究

• 批准号: 13308041
• 负责人: HANAOKA Fumio
• 金额: $ 30.28万
• 依托单位: OSAKA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13308041/
• 关键词: nucleotide excision repair; translesion synthesis; XPC protein; HR23B protein; xeroderma pigmentosum; thymine DNA glycosylase; DNA polymerase η; DNA polymerase τ; SUMO-1; ユビキチン; DDB; 色素乾皮症; APエンドヌクレアーゼ; SUMO-I

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.

为了研究DNA修复系统的调控机制，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，得到以下结果：1）XPC-HR 23 B蛋白复合物是全基因组NER的起始蛋白，它与一个中心体蛋白centrin 2.2）XPC-HR 23 B强烈识别DNA的特异二级结构，涉及单链和双链连接。使用环状DNA底物的DNA酶I足迹分析显示，单个XPC-HR 23 B复合物以不对称的方式保护扭曲的位点。3）虽然mHR 23 A KO小鼠未显示异常，但mHR 23 B KO小鼠显示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）酵母双杂交筛选发现XPC与胸腺嘧啶DNA糖基化酶（TDG）相互作用。通过生化分析，XPC与TDG不仅在物理上相互作用，而且在功能上也相互作用。5）我们鉴定了芽殖酵母Rad 4和人XPC的两个裂殖酵母同源物，命名为Rfp 4A和Rhp 4 B。6）人DNA聚合酶η（Pol η）催化5 R-胸腺嘧啶二醇和5S-胸腺嘧啶二醇的TLS反应相对高效和准确。7）在129个小鼠品系中发现DNA聚合酶τ（Pol τ）基因的两个等位基因均存在无效突变。Pol τ缺陷小鼠的总体突变频率和谱是正常的。

项目成果

Sugasawa, K., Shimizu, Y., Iwai, S., Hanaoka, F.: "A molecular mechanism for DNA damage recognition by the xeroderma pigmentosum group C protein complex."DNA Repair. 1. 95-107 (2002)

Sugasawa, K.、Shimizu, Y.、Iwai, S.、Hanaoka, F.：“着色性干皮病 C 组蛋白复合物识别 DNA 损伤的分子机制。”DNA 修复。

Fujiwara, K. et al.: "Structure of the ubiquitin-interacting motif of S5a bound to the ubiquitin-like domain of HR23B"J.Biol.Chem.. 279. 4760-4767 (2004)

Fujiwara, K. 等人：“与 HR23B 泛素样结构域结合的 S5a 泛素相互作用基序的结构”J.Biol.Chem.. 279. 4760-4767 (2004)

Kuraoka, I. et al.: "Oxygen free-radical damage to DNA polymenas η and resistanteto exonaclease action at cyclopurine deoxynucleaside residues"J. Biol. Chem.. 276. 49283-49288 (2001)

Kuraoka, I. 等人：“氧自由基对 DNA 多聚酶的损伤和对环嘌呤脱氧核苷残基的外切酶作用的抵抗”J. Biol. 276. 49283-49288 (2001)

Araki, M. et al.: "Centrosome protein centrin/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair"J.Biol.Chem.. 276. 18665-18672 (2001)

Araki, M. 等人：“中心体蛋白 centrin/caltractin 1 是着色性干皮病 C 组复合体的一部分，它启动全基因组核苷酸切除修复”J.Biol.Chem.. 276. 18665-18672 (2001)

Fukumoto, Y. et al.: "Two budding yeast RAD4 homologs in fission yeast play different roles in the repair of UV-induced DNA damage"DNA Repair. 1. 833-845 (2002)

Fukumoto, Y. 等人：“裂殖酵母中的两种出芽酵母 RAD4 同源物在修复紫外线诱导的 DNA 损伤中发挥不同的作用”DNA 修复。