HYBRID ENDONUCLEASES FOR GENOMIC RESEARCH AND ANALYSIS

用于基因组研究和分析的混合核酸内切酶

• 批准号: 2910211
• 负责人: SRINIVASAN CHANDRASEGARAN
• 金额: $ 18.95万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910211
• 关键词: DNA binding protein; biomedical resource; chimeric proteins; conformation; genetic library; genome; health science research; high performance liquid chromatography; hybrid enzyme; molecular cloning; nuclear magnetic resonance spectroscopy; oligonucleotides; polymerase chain reaction; protein engineering; protein structure; restriction endonucleases

A long term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence-specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of brute-force screening of bacteria and other microorganisms for new enzymes. Our objective here is to generate rare cutters that would be valuable tools in genomic research and analysis. Over the past six years, we have studied FokI restriction endonuclease from Flavobacterium Okenokoites in great detail. FokI is a Type IIS endonuclease which recognizes the pentanucleotide duplex, 5'-GGATG-3': 5'-CATCC-3' and cleaves about 9/13 bp away from the recognition site. This implies the presence of two separate protein domains in this enzyme: one for sequence-specific recognition and the other for the endonuclease activity. Our studies o proteolytic fragments of FokI endonuclease have defined an N-terminal DNA- binding domain and C-terminal domain with non-specific cleavage activity (PNAS 89:4275-4279(1991)]. These results have been confirmed by the study of the C-terminal deletion mutants of FokI endonuclease [Gene 133:79-84 (1993)]. Furthermore, introduction of additional amino acid residues between the recognition and cleavage domains of FokI can alter athe cleavage distance from the recognition site within its DNA substrate [PNAS 90:2764-2768 (1993); J. Biol Chem.269:31978-31982 (1994)]. These results suggest that the two domains of FokI are connected by a liner region which appears to be amenable for repositioning of the DNA-binding domain with respect to the catalytic domain. Recently, we have successfully engineered th first chimeric restriction endonuclease by linking the Ubx homeodomain to the catalytic domain of (Fn) of FokI [PNAS 91:883-887 (1994)]. More recently, we reported the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of FokI endonuclease. Both fusions are active. This opens the way to generate many new enzymes with tailor-made sequence-specificities [Science, manuscript submitted for publication (1995)]. This work could lead to the development of an array of artificial nucleases with tailor- made sequence-specificities desirable for various applications.

在限制修改酶领域的长期目标已经是 通过新序列特异性产生限制性核酸内切酶 突变或工程现有酶。 这将避免越来越多的 细菌和其他微生物的蛮力筛查的艰巨任务 用于新酶。 我们的目标是生成稀有的切割机 在基因组研究和分析中成为有价值的工具。 在过去的六个 多年，我们研究了黄杆菌的FOKI限制性核酸内切酶 Okenokoites非常详细。 Foki是IIS类核酸内切酶 识别五核苷酸双工，5'-ggatg-3'：5'-catcc-3'和Cleaves 距识别地点约9/13 bp。 这意味着存在 该酶中的两个单独的蛋白质结构域：一个用于序列特异性的 识别性，另一个用于核酸内切酶活性。 我们的研究o foki核酸内切酶的蛋白水解片段已定义了N端DNA- 具有非特异性切割活性的结合结构域和C末端结构域 （PNAS 89：4275-4279（1991）。 FOKI核酸内切酶的C末端缺失突变体[Gene 133：79-84 （1993）]。 此外，引入其他氨基酸残基 在Foki的识别和裂解域之间可以改变ATHE DNA底物内识别位点的切割距离[PNAS 90：2764-2768（1993）; J. Biol Chem.269：31978-31982（1994）]。 这些结果 建议FOKI的两个结构域通过衬里区域连接 似乎适合重新定位DNA结合域 尊重催化域。 最近，我们已经成功地设计了 第一个嵌合限制性核酸内切酶通过链接UBX同源域 到FOKI（FN）的催化结构域[PNAS 91：883-887（1994）]。 更多的 最近，我们报道了新颖特定地点的故意创建 通过将两种不同的锌指蛋白与乳沟联系起来来进行核酸 FOKI核酸内切酶的域。 两种融合都是活跃的。 这打开了 用量身定制的序列特异性生成许多新酶 [科学，手稿提交出版（1995）]。 这项工作可以 导致通过量身定制的一系列人造核酸酶的发展 对于各种应用来说，都需要进行序列特异性。