Functional analysis of the hinge region close to the PCNA-binding region of human FEN1

人 FEN1 靠近 PCNA 结合区的铰链区的功能分析

• 批准号: 18590046
• 负责人: MORIOKA Hiroshi
• 金额: $ 2.59万
• 依托单位: Kumamoto University (2007)Hokkaido University (2006)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590046/
• 关键词: FEN1; PCNA; DNA Replication; DNA Repair; 5'-flap DNA; Biomolecular Interactions; Domain Structure; Flap構造; 構造特異的ヌクレアーゼ; 酵素活性促進機構; 5' Flap DNA; ヒンジ領域; NaCl濃度依存性

Flap endonuclease 1(FEN1) and proliferating cell nuclear antigen (PCNA) play a crucial role in DNA replication and repair. FEN1 is a structure-specific endonuclease that recognizes a branched DNA structure consisting of a double-stranded DNA with 5'-unannealed flap(5'-flap DNA). PCNA acts as a DNA clamp protein and binds to FEN1 on 5'-flap DNA, then stimulates its nuclease activity. The structural basis of the human FEN1-PCNA interaction was revealed by analysis of the crystal structure of the complex between human FEN1 and PCNA. FEN1 possesses a short linker region containing small residues (-^<333>QGST^<336>-) between the core domain and the PCNA-interacting protein(PIP) motif in the C-terminal tail, and this linker region acts as a hinge, which may play a role in switching its nuclease activity. In order to investigate the importance of this hinge region in terms of the nuclease activity of FEN1 bound to PCNA by assaying the activity of PCNA to stimulate the nuclease activity of FEN … More 1, we performed mutation experiments against this hinge region of FEN1.The human FEN1 mutants with longer and flexible sequences in the linker region (-GSGS-(GS2), -GSGSGS-(GS3), -GSGSGSGS-(GS4), -GSGSGSGSGS-(GS5))were prepared, and flap endonuclease assays and FEN1-PCNA binding assays were performed. Extension of more than two linker residues (GS3, GS4, GS5)resulted in decreased stimulation by PCNA, while every mutant showed the same nuclease activity as wild type without PCNA. These results clearly indicate the importance of the length of hinge region in terms of stimulation by PCNA to direct the FEN1 core domain toward the DNA substrate. Furthermore, the relationship between the nuclease activity and NaCl concentration was investigated using PCNA mutants, since it was known that FEN1 activity was highly influenced by salt concentration. Results of kinetic measurements of nuclease activity of FEN1 suggested that there were significant correlations between the PCNA/FEN1 complex formation and the NaCl concentration. Less

FEN1和增殖细胞核抗原在DNA复制和修复中起着至关重要的作用。FEN1是一种结构特异性内切酶，它识别由带有5‘-未退火瓣的双链DNA(5’-瓣DNA)组成的分枝DNA结构。增殖细胞核抗原作为DNA钳制蛋白与FEN1在5‘-FAP DNA上结合，进而刺激其核酸酶活性。通过对人FEN1与增殖细胞核抗原复合物晶体结构的分析，揭示了人FEN1与增殖细胞核抗原相互作用的结构基础。FEN1在核心区和C末端的增殖细胞核抗原相互作用蛋白(PIP)基序之间有一个含有少量残基(-^&lt；333&gt；QGST^&lt；336&gt；-)的短连接区，该连接区作为一个铰链，可能在其核酸酶活性的转换中发挥作用。为了通过检测增殖细胞核抗原刺激Fen…核酸酶活性的方法，探讨该铰链区在FEN1与增殖细胞核抗原结合的核酸酶活性中的重要性在此基础上，我们针对FEN1铰链区进行了突变实验，制备了FEN1链接区具有较长和灵活序列的人FEN1突变体(-GSGS-(GS2)、-GSGSGS-(GS3)、-GSGSGSGS-(GS4)、-GSGSGSGS-(GS5))，并进行了FEN1-增殖细胞核抗原结合试验和FEN1-FEN1-增殖细胞核抗原结合试验。超过两个连接子残基(GS3、GS4、GS5)的延伸导致了增殖细胞核抗原刺激作用的减弱，而每个突变体都表现出与野生型相同的核酸酶活性。这些结果清楚地表明了铰链区的长度在增殖细胞核抗原刺激下将FEN1核心区导向DNA底物的重要性。此外，由于已知FEN1的活性受盐浓度的很大影响，我们还利用增殖细胞核抗原突变体研究了核酸酶活性与盐浓度的关系。FEN1核酸酶活性的动力学测定结果表明，增殖细胞核抗原/FEN1复合体的形成与盐浓度有显著的相关性。较少

项目成果

Molecular docking of porphyrins with cationic limbs on intramolecular G-quadruplex.

卟啉与分子内 G-四链体上的阳离子肢的分子对接。

• 发表时间: 2007
• 期刊: Nucleic Acids Symp. Ser. 51
• 作者: Ishikawa;Y.;Higashi;E.;Morioka;H
• 通讯作者: H

Crystallization and preliminary crystallographic analysis of the catalytic domain of human flap endonuclease 1 in complex with a nicked DNA product:use of a DPCS kit for efficient protein-DNA complex crystallization.

与带切口 DNA 产物复合的人瓣核酸内切酶 1 催化结构域的结晶和初步晶体学分析：使用 DPCS 试剂盒进行高效的蛋白质-DNA 复合物结晶。

• 发表时间: 2008
• 期刊: Acta Crystallogr. F 64
• 作者: Sakurai;S.;Kitano;K.;Morioka;H. & Hakoshima;T.
• 通讯作者: T.

Crystallization and preliminary crystallographic analysis of the catalytic domain of human flap endonuclease 1 in complex with a nicked DNA product : use of a DPCS kit f6r efficient protein-DNA complex crystallization

与带切口 DNA 产物复合的人瓣核酸内切酶 1 催化结构域的结晶和初步晶体学分析：使用 DPCS 试剂盒进行高效蛋白质-DNA 复合物结晶

• 发表时间: 2008
• 期刊: Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun 64 (Pt 1)
• 作者: Sakurai;S.;Kitano;K.;Morioka;H.;Hakoshima;T
• 通讯作者: T

Biochemical and biological properties of DNA photolyases derived from ultraviolet-sensitive rice cultivars.

来自紫外线敏感水稻品种的 DNA 光裂合酶的生化和生物学特性。

• 发表时间: 2007
• 期刊: Genes Genet. Syst. 82
• 作者: Wan-Ling Hsu.;et al.;Y. Ishikawa;Y. Ishikawa;Y. Ishikawa;石川 裕二;伊藤真之;伊藤真之;Masayuki Itoh;伊藤真之;伊藤真之;関西ネットワークシステム編;Fedina I.;Okafuji A.;Sudo E;Hidema J.;A. Yamamoto;Y. Iwamatsu;M. Teranishi;寺西美佳;山岸朋香;A. Yamamoto;Y. Iwamatsu;M. Teranishi;J. Hidema;A. Yamamoto
• 通讯作者: A. Yamamoto

Molecular docking of porphyrins with cationic limbs on intramolecular G-quadruplex

卟啉与分子内 G-四链体上的阳离子肢的分子对接

• 发表时间: 2007
• 期刊: Nucleic Acids Symp.Ser. 51
• 作者: Ishikawa;Y.
• 通讯作者: Y.