Chemical Creation of Artificial Restriction Enzyme and Its Application to Genomic Analysis

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The molecular mechanism of bleomycin antibiotics facilitated design and synthesis of srtificial DNA cleaving molecules. Transformation of a sequence-specific DNA-binding molecule into a sequence-specific DNA-cleaving molecule allows identification of the preferred binding locations of these molecules on restriction fragments analyzed on sequencing gels. Various types of DNA-cleaving molecules have been demonstrated. Sequence-specific DNA-cleaving peptides and oligonucleotides equipped with a DNA-cleaving moiety have also been designed. Zinc finge motif is a novel DNA binding motif characterized by the unique role of zinc ; the protein folding and the DNA binding ability are goverened by the coordination of a zinc ion. In C_2H_2-type zinc finger, two cysteines and two histidines contribute to form a globular domain through zinc coordination. The zinc finger of C_2H_2-type gives promise of recognition for any DNA sequences because of its recognition mode. A C_2H_2-type zinc finger protein Sp1 has been converted into artificial sitespecific nuclease with an attached Ni-based DNA cleavage unit (Gly-Gly-His). This protein cleaved DNA at a single site near the Sp1 recognition sequence. The zinc finger-based nuclease is applicable to chromosome mapping and sequencing.