Mechanism of DNA excision repair based on the three-dimensional structure of UvrABC endonuclease

基于UvrABC核酸内切酶三维结构的DNA切除修复机制

• 批准号: 12680659
• 负责人: FUKUYAMA Keiichi
• 金额: $ 2.24万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680659/
• 关键词: exonuclease; RecJ; DNA repair; thermophilic bacterium; overexpression; ドーパミン; ノルアドレナリン; 電子伝達系; チトクロムb_<561>; 電子伝達; ビタミンC; 膜タンパク質

RecJ is a 5' to 3' exonuclease specific for single-stranded DNA, and involved in DNA repair and recombination systems. Recently, RecJ has been highlighted as a key enzyme for the replication-recombination relationship. RecJ homologues have five characteristic motifs, which form a large family of the predicted phosphoesterases, named by DHH family.We performed the X-ray crystallography of RecJ from Thermus thermohilus HB8 (ttRecJ).As ttRecJ was expressed as an inclusion body, it was purified through denaturation and refolding. The region of the catalytic core domain (cd-ttRecJ) was identified by limited proteolysis. Cd-ttRecJ, expressed as soluble form, was highly purified and crystallized.The structure of RecJ was solved by single-wavelength anomalous dispersion, using Se-Met cd-ttRecJ. RecJ has a novel fold, in which two domains are interconnected with a long helix to form a central groove. This groove is composed of conserved residues and positively charged, which may be involved in DNA binding. The metal ion is coordinated by the motifs characteristic in DHH family. This metal ion was identified as Mn^<2+> by an anomalous scattering experiment.

RecJ 是一种单链 DNA 特异性的 5' 至 3' 核酸外切酶，参与 DNA 修复和重组系统。最近，RecJ 被认为是复制重组关系的关键酶。 RecJ同源物有5个特征基序，形成了预测的磷酸酯酶大家族，命名为DHH家族。我们对Thermus thermohilus HB8 (ttRecJ)的RecJ进行了X射线晶体学分析。由于ttRecJ以包涵体形式表达，因此通过变性和重折叠纯化。通过有限的蛋白水解鉴定催化核心结构域（cd-ttRecJ）的区域。 Cd-ttRecJ以可溶形式表达，经过高度纯化和结晶。利用Se-Met cd-ttRecJ，通过单波长反常色散解析了RecJ的结构。 RecJ具有一种新颖的折叠，其中两个结构域通过长螺旋相互连接以形成中心凹槽。该凹槽由保守残基组成，带正电荷，可能参与 DNA 结合。金属离子由 DHH 家族特征的基序配位。通过反常散射实验将该金属离子鉴定为Mn 2+ 。

项目成果

Takeuchi, K.: "Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy : comparison with the putidaredoxin-cytochrome P450cam system"J. Biochem.. 130. 789-797 (2001)

Takeuchi, K.：“EPR 光谱揭示的肾上腺氧还蛋白-细胞色素 P450scc 相互作用：与 putidaredoxin-细胞色素 P450cam 系统的比较”J.

Asada, A.: "Planarian cytochrome b561 : Conservation of a six transmembrane structure and localization along the central and peripheral nervous system"J. Biochem.. 131. 175-182 (2002)

Asada, A.：“涡虫细胞色素 b561：六跨膜结构的保守性以及沿中枢和周围神经系统的定位”J.

T.subaki,M.: "Diethylpyro carbonate-modification abolishes fast electron accepting ability of cytodirome b561 from ascorbate but does not influence on electron donation to moncdehydroascor hate radical"Biochemistry. 39. 3276-3284 (2000)

T.subaki,M.：“二乙基焦碳酸酯修饰消除了抗坏血酸的细胞二组 b561 的快速电子接受能力，但不影响向单脱氢抗坏血酸自由基的电子供给”生物化学。

Neya,S.: "Unusual spin state equilibrium of azide metmyoglobin induced by core de formed heme."Inorg.Chem.. (in press). (2001)

Neya，S.：“由核心形成的血红素诱导的叠氮化物高铁肌红蛋白的异常自旋态平衡。”Inorg.Chem..（印刷中）。

Neya, S.: "Unusual spin state equilibrium of azide metmyoglobbin induced by core deformed heme"Inorg. Chem.. 40. 1220-1225 (2001)

Neya，S.：“核心变形血红素诱导的叠氮化物肌红蛋白的异常自旋态平衡”Inorg。