| Budget Amount *help |
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2000: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1999: ¥12,200,000 (Direct Cost: ¥12,200,000)
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| Research Abstract |
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.
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