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A STUDY ON DNA CLEAVAGE MECHANISM BY WATER-SOLUBLE PORPHYRINS

水溶性卟啉DNA切割机制的研究

基本信息

批准号：
11470476
负责人：
UNO Tadayuki
金额：
$ 9.6万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The purpose of this project is to design and synthesize new water-soluble porphyrins and to develop DNA-cleaving reagents through investigation of their scission activity for various nucleotide sequences. To enhance the sequence specificity, we firstly synthesized porphyrin derivatives bearing an acridine moiety which has high affinity to GC base pairs. The derivatives were highly soluble in water, and showed high affinity to DNA, as revealed by visible absorption spectroscopy. We synthesized some derivatives with variable linker length between acridine and the porphyrin moieties, and studied their binding affinity to DNA.These derivatives were found to intercalate between the DNA base pairs with the acridine moiety, the porphyrin moiety being located to the solvent side. In addition, a derivative with six hydrocarbons as a linker showed the highest activity in photocleavage of DNA.Some proteins bind to DNA with nucleotide-specific manner, and an iron complex of porphyrin (heme) activates oxygen. Based on these characters, we tried to develop oxidative DNA-cleaving heme proteins. We cloned a heme-containing transcription activator, CooA, by totally synthesizing the gene in vitro, and constructed an over-expression system of this protein. In addition, we prepared some site-directed mutants of a four-helix bundle protein, cytochrome b562. Since this protein has a six-coordinated heme, we first constructed a heme pocket, which is a pre-requisite to activate oxygen. The heme was found to escape from the protein matrix when one of the axial ligand, Met7, was replaced by Gly. Thus, we further replaced two Glu residues at position 4 and 8 by Ser simultaneously, and stabilized the heme. To achieve higher oxidation activity, the other axial ligand, His102, was replaced by Cys. This mutant retained the heme, and the Cys remained the ligand even in the ferrous state.

本课题旨在通过研究水溶性卟啉对不同核苷酸序列的裂解活性，设计合成新型水溶性卟啉，并开发dna裂解试剂。为了提高序列特异性，我们首先合成了含有与GC碱基对具有高亲和力的吖啶片段的卟啉衍生物。可见吸收光谱分析表明，该衍生物具有高水溶性，对DNA具有高亲和力。我们合成了一些在吖啶和卟啉基团之间具有可变连接长度的衍生物，并研究了它们与DNA的结合亲和力。这些衍生物被发现插入DNA碱基对之间的吖啶部分，卟啉部分位于溶剂侧。此外，以六种碳氢化合物为连接物的衍生物在DNA的光裂解中表现出最高的活性。一些蛋白质以核苷酸特异性的方式与DNA结合，卟啉（血红素）的铁复合物激活氧气。基于这些特性，我们尝试开发氧化dna切割血红素蛋白。我们通过体外完全合成克隆了一个含血红素的转录激活子CooA，并构建了该蛋白的过表达体系。此外，我们还制备了一些四螺旋束蛋白细胞色素b562的定点突变体。由于这种蛋白质具有六配位血红素，我们首先构建了一个血红素口袋，这是激活氧气的先决条件。当其中一个轴向配体Met7被Gly取代时，血红素从蛋白质基质中逸出。因此，我们进一步用Ser同时取代了位于4和8位的两个Glu残基，并稳定了血红素。为了获得更高的氧化活性，另一个轴向配体His102被Cys取代。这个突变体保留了血红素，而Cys甚至在亚铁状态下仍然是配体。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Tadayuki Uno: "Cysteine Thiolate Coordination in the Ferrous CO Complex of an Engineered Cytochrome b562"J.Am.Chem.Soc.. 123. 2458-2459 (2001)

Tadayuki Uno：“工程化细胞色素 b562 的亚铁 CO 复合物中的半胱氨酸硫醇盐配位”J.Am.Chem.Soc.. 123. 2458-2459 (2001)

DOI：
发表时间：
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0
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通讯作者：

Yoshinobu Ishikawa, Aya Yamashita, and Tadayuki Uno: "Efficient photocleavage of DNA by cationic porphyrin-acridine hybrids with the effective length of diamino alkyl linkage"Chem.Pharm.Bull.. 49. 287-293 (2001)

Yoshinobu Ishikawa、Aya Yamashita 和 Tadayuki Uno：“通过具有有效长度的二氨基烷基键的阳离子卟啉-吖啶杂化物对 DNA 进行有效的光裂解”Chem.Pharm.Bull.. 49. 287-293 (2001)

DOI：
发表时间：
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影响因子：
0
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通讯作者：

Yoshinobu Ishikawa: "Efficient photocleavage of DNA by cationic porphyrin-acridine hybrids with the effective length of diamino alkyl linkage"Chem.Pharm.Bull.. 49(印刷中). (2001)

Yoshinobu Ishikawa：“通过具有有效长度的二氨基烷基键的阳离子卟啉-吖啶杂化物对 DNA 进行有效的光裂解”Chem.Pharm.Bull.. 49（出版中）。

DOI：
发表时间：
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影响因子：
0
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通讯作者：

Tadayuki Uno, Akihiro Yukinari, Yoko Moriyama, Yoshinobu Ishikawa, Yoshikazu Tomisugi, James A.Brannigan, and Anthony J.Wilkinson: "Engineering a Ligand Binding Heme Pocket into a Four Helix Bundle Protein Cytochrome b562"J.Am.Chem.Soc.. 123. 512-513 (200

Tadayuki Uno、Akihiro Yukinari、Yoko Moriyama、Yoshinobu Ishikawa、Yoshikazu Tomisugi、James A.Brannigan 和 Anthony J.Wilkinson：“将配体结合血红素口袋设计成四螺旋束蛋白细胞色素 b562”J.Am.Chem.Soc。