A STUDY ON DNA CLEAVAGE MECHANISM BY WATER-SOLUBLE PORPHYRINS

本计画的目的是设计合成新的水溶性卟啉化合物，并借由研究其对不同核苷酸序列的切割活性，开发DNA切割试剂。为了增强序列特异性，我们首先合成了带有吖啶基团的卟啉衍生物，该基团对GC碱基对具有高亲和力。衍生物是高度可溶于水，并显示出高亲和力的DNA，如可见吸收光谱所揭示的。我们合成了一些吖啶-卟啉连接基长度可变的衍生物，并研究了它们与DNA的结合能力，发现这些衍生物的吖啶部分插入DNA碱基对之间，卟啉部分位于溶剂侧。此外，以六种碳氢化合物为连接基团的卟啉衍生物对DNA的光裂解活性最高，某些蛋白质以核苷酸特异性的方式与DNA结合，卟啉（血红素）的铁络合物可活化氧。基于这些特性，我们试图开发氧化切割DNA的血红素蛋白。我们通过体外全合成的方法克隆了一个含血红素的转录激活因子CooA，并构建了该蛋白的过表达系统。此外，我们制备了一些定点突变的四螺旋束蛋白，细胞色素b562。由于这种蛋白质有一个六配位的血红素，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。血红素被发现逃离蛋白质基质时，轴向配体之一，蛋氨酸7，被替换为甘氨酸。因此，我们进一步用Ser同时替换了位置4和8处的两个Glu残基，并且稳定了血红素。为了实现更高的氧化活性，另一个轴向配体His 102被Cys取代。该突变体保留了血红素，并且半胱氨酸即使在亚铁状态下也仍然是配体。

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.