Structural Basis for mechanisms of preventing genomic instability

预防基因组不稳定机制的结构基础

• 批准号: 15390016
• 负责人: YAMAGATA Yuriko
• 金额: $ 9.22万
• 依托单位: KUMAMOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390016/
• 关键词: Preventing genomic instability; Enzyme-substrate interaction; Protein Crystallography; Structural biology; Bio-molecular interaction; 蛋白質-リガンド相互作用; DNA複製; 蛋白質X線結晶構造解析

Cellular DNAs, RNAs and Nucleotides are remarkable instable. Therefore cells have varieties of preventing systems for genomic instability. In this study, we target the proteins which work during pre-, first or middle stage of DNA replication. Our research purpose is to elucidate the preventing mechanisms for the genomic instability at the atomic level by the determinations of these protein structures.The structures of MutT and hMTH1 were determined in the complexes with substrates and products by X-ray crystallography. The crystal structures of MutT alone, and in complex with a product, 8-oxo-dGMP(MutT-8-oxo-dGMP) have shown that MutT specifically recognizes 8-oxo-dGMP through a wealth of hydrogen bonds to the protein and waters in the binding pocket with the large ligand-induced conformational change. The mammalian counterpart of MutT, MutT homolog-1(MTH1), can hydrolyze a variety of nucleoside triphosphates containing oxidized adenine. The structural basis for the difference of the substrate specificity between MutT and hMTH1 is of fundamental interest. The crystal structure of MTH1 complexed with a product, 8-oxo-dGMP(hMTH1-8-oxo-dGMP) shows that the means of 8-oxoG recognition by hMTH1 are different from those found in the structure of MutT-8-oxo-dGMP. The structure of the substrate-binding pocket in hMTH1-8-oxo-dGMP suggests that hMTH1 can recognize several oxidatively damaged purine nucleoside triphosphates without a large conformational change.The crystals of N-terminally His-tagged NUDT5 grew, but the resolution of the diffraction was relatively low. Now we are trying to get the crystals diffracting to a higher resolution by cutting the His-Tag.The crystallization trials of purified primase and AlkB were in progress.

细胞DNA、RNA和核苷酸是显著不稳定的。因此，细胞具有多种防止基因组不稳定性的系统。在这项研究中，我们的目标是在DNA复制的前，第一或中期阶段的蛋白质。我们的研究目的是通过对这些蛋白质结构的测定，在原子水平上阐明防止基因组不稳定性的机制。MutT单独以及与产物8-氧代-dGMP（MutT-8-氧代-dGMP）复合的晶体结构表明，MutT通过与蛋白质和结合口袋中的沃茨的大量氢键特异性识别8-氧代-dGMP，具有大的配体诱导的构象变化。MutT的哺乳动物对应物MutT同系物-1（MTH 1）可以水解多种含有氧化腺嘌呤的核苷三磷酸。MutT和hMTH 1之间底物特异性差异的结构基础具有根本意义。MTH 1与产物8-oxo-dGMP（hMTH 1 -8-oxo-dGMP）复合的晶体结构表明，hMTH 1识别8-oxoG的方式与MutT-8-oxo-dGMP结构中发现的方式不同。hMTH 1 -8-oxo-dGMP中底物结合口袋的结构表明hMTH 1可以识别几种氧化损伤的嘌呤核苷三磷酸而没有大的构象变化。N端His标记的NUDT 5晶体生长，但衍射分辨率相对较低。目前，我们正在尝试通过切割His标签来获得更高分辨率的晶体，纯化的引发酶和AlkB的结晶试验正在进行中。

项目成果

タンパク質のかたちから生命の謎を解く

从蛋白质的形状解开生命之谜

• 发表时间: 2005
• 作者: Lee S.J.et al.;Tsunaka Y. et al.;Yoshioka Y. et al.;Hioki Y. et al.;Mishima M. et al.;Nakamura T. et al.;Shibata H. et al.;田之倉優編;田之倉優 編
• 通讯作者: 田之倉優 編

Structure of human MTH1, a nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates

• DOI: 10.1074/jbc.m402393200
• 发表时间: 2004-08-06
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Mishima, M;Sakai, Y;Shirakawa, M
• 通讯作者: Shirakawa, M

Functionalization of TNF-alpha using phage display technique and PEGylation improves its antitumor therapeutic window.

使用噬菌体展示技术和聚乙二醇化对 TNF-α 进行功能化可改善其抗肿瘤治疗窗口。

• 发表时间: 2004
• 期刊: Clin. Cancer Res. 10・24
• 作者: Shibata H.;et al.
• 通讯作者: et al.

Crystallization and preliminary X-ray analysis of Escherichia coli MutT in binary and ternary complex forms.

二元和三元复合物形式的大肠杆菌 MutT 的结晶和初步 X 射线分析。

• 发表时间: 2004
• 期刊: Acta Cryst. D60
• 作者: Nakamura;T.;Doi;T.;Sekiguchi;M.;Yamagata;Y.
• 通讯作者: Y.

Takano K. et al.: "Buried Water Molecules Contribute to the Conformational Stability of a Protein"Protein Engineering. 16. 5-9 (2003)

Takano K. 等人：“埋藏的水分子有助于蛋白质的构象稳定性”蛋白质工程。