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

人工限制性内切酶的化学合成及其在基因组分析中的应用

• 批准号: 05557111
• 负责人: SUGIURA Yukio
• 金额: $ 9.73万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05557111/
• 关键词: Artificial Restriction Enzymo; Bleomycin; DNA Cutting; Zinc Finger; Nuclease; Triple Stranded DNA; Human Geome; ヒトゲノム; 塩基配列特異性; エンジイン; DNA結合蛋白質; 人工リプレッサー; 転写因子; 活性酸素; ニッケル錯体; グリシル・グリシル・ヒスチジン

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.

博来霉素抗生素的分子机制为人工DNA切割分子的设计和合成提供了便利。将序列特异性dna结合分子转化为序列特异性dna切割分子，可以确定这些分子在测序凝胶上分析的限制性内切片段上的首选结合位置。各种类型的dna切割分子已经被证明。序列特异性dna切割肽和配备dna切割片段的寡核苷酸也被设计。锌指基序是一种以锌的独特作用为特征的新型DNA结合基序；蛋白质的折叠和DNA的结合能力是由锌离子的配位控制的。在c_2h_2型锌指中，两种半胱氨酸和两种组氨酸通过锌配位形成球状结构域。由于c_2h_2型锌指的识别模式，它对任何DNA序列都有识别的希望。将一种c_2h_2型锌指蛋白Sp1转化为带有ni基DNA裂解单元（Gly-Gly-His）的人工位点特异性核酸酶。该蛋白在Sp1识别序列附近的单个位点切割DNA。锌指核酸酶适用于染色体定位和测序。

项目成果

J. Kuwahara: "Binding of transcription factor Sp1 to GC box revealed by footprinting analysis" Biochemistry. 32. 5994-6001 (1993)

J. Kuwahara：“通过足迹分析揭示转录因子 Sp1 与 GC 盒的结合”生物化学。

M.Nagaoka: "A novel zinc finger-based DNA cutter" J.Am.Chem.Soc.116. 4085-4086 (1994)

M.Nagaoka：“一种新型的基于锌指的 DNA 切割器”J.Am.Chem.Soc.116。

J.Kuwahara: "Molecular design of a zinc fingertype DNA cutter" Protein, Nucleic Acids, and Enzyme. 40. 1457-1464 (1995)

J.Kuwahara：“锌指型 DNA 切割器的分子设计”蛋白质、核酸和酶。

M.Nagaoka: "A novel zinc finger-based DNA cutter:Biosynthetic design and highly selective DNA cleavage" J.Am.Chem.Soc.116. 4085-4086 (1994)

M.Nagaoka：“一种新型的基于锌指的 DNA 切割器：生物合成设计和高选择性 DNA 切割”J.Am.Chem.Soc.116。

杉浦幸雄: "長鎖DNAの化学切断" 蛋白質 核酸 酵素. 38. 516-523 (1993)

Yukio Sugiura：“长链 DNA 的化学裂解”蛋白质核酸酶 38. 516-523 (1993)