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

博莱霉素类抗生素的分子机制促进了人工DNA裂解分子的设计和合成。将序列特异的DNA结合分子转化为序列特异的DNA裂解分子，可以在测序凝胶上分析的限制性片段上鉴定这些分子的首选结合位置。已经证明了各种类型的DNA裂解分子。还设计了序列特异的DNA裂解多肽和配备DNA裂解部分的寡核苷酸。锌粉基序是一种新的DNA结合基序，具有锌的独特作用，蛋白质的折叠和DNA结合能力是由锌离子的配位控制的。在C_2H_2型锌指中，两个半胱氨酸和两个组氨酸通过锌配位形成球状结构域。C_2H_2型锌指由于其识别模式，有望识别任何DNA序列。将C_2H_2型锌指蛋白Sp1转化为含镍基链DNA裂解单元(Gly-Gly-His)的人工位点特异性核酸酶。这种蛋白质在Sp1识别序列附近的单一位置切割DNA。该锌指核酸酶适用于染色体定位和测序。

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.