STRUCTURAL STUDIES OF INTRON ENCODED ENDONUCLEASES

内含子编码核酸内切酶的结构研究

• 批准号: 2910377
• 负责人: PATRICK M VAN ROEY
• 金额: $ 19.87万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-05 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910377
• 关键词: Archaea; X ray crystallography; active sites; bacteriophage T4; endodeoxyribonuclease; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate complex; introns; mutant; nuclear magnetic resonance spectroscopy; posttranslational modifications; protein structure function; thymidylate synthase

The goal of this application is to study the mechanism of action and the substrate recognition properties of two intron-encoded endonucleases, I-DmoI and I-TevI, that catalyze double-strand DNA cleavage to initiate mobility of their introns. These endonucleases are remarkable in that they interact with lengthy recognition sequences. They possess exquisite cleavage site specificities and distort their DNA substrates. I-DmoI and I-TevI represent the two major subclasses of homing endonucleases. I-DmoI is a member of the majority class, containing two copies of a conserved motif, LAGLI-DADG. This enzyme recognizes a 14 base pair binding site in a sequence-specific manner, cleaves close to the intron insertion site and leaves a 4-nucleotide 3'-overhang. I- TevI, a member of the GIY-YIG family, recognizes a 35 base pair DNA target in a sequence-tolerant manner, cleaves 23 to 25 nucleotides upstream of the intron insertion site and leaves 2-nucleotide 3'- overhangs. While LAGLI-DADG enzymes interact with their substrates mainly through the major groove of the DNA, I-TevI, the best characterized of the GIY-YIG enzymes, interacts predominantly through the minor groove. The specific aims of the project are to determine the three-dimensional structures of the enzymes, of isolated domains and of complexes with substrates by a combination of X-ray crystallography and NMR spectroscopy. This will allow the comparison of representative examples of the two classes of enzymes that, although otherwise unrelated, carry out the same biological function.

本申请的目的是研究作用机制和药物的生物学活性。 两种内含子编码的核酸内切酶的底物识别特性， I-DmoI和I-TevI，催化双链DNA切割以启动 内含子的移动性。 这些核酸内切酶的显著之处在于， 它们与长的识别序列相互作用。 他们拥有 精确的切割位点特异性并扭曲它们的DNA底物。 I-DmoI和I-TevI代表归巢的两个主要子类 核酸内切酶。I-DmoI是多数类的成员，包含两个 保守基序LAGLI-DADG的拷贝。 这种酶识别14 碱基对结合位点，切割接近 内含子插入位点并留下4个核苷酸的3 '突出端。 我... TevI是GIY-YIG家族的成员，识别35个碱基对的DNA 以序列耐受方式靶向，切割23至25个核苷酸 内含子插入位点的上游，并留下2个核苷酸3 '- 悬垂部分。 虽然LAGLI-DADG酶与其底物相互作用， 主要是通过DNA的大沟，I-TevI，最好的 特征为GIY-YIG酶，主要通过 小沟 该项目的具体目标是确定三维 酶的结构，分离的结构域和与 通过X射线晶体学和NMR的组合， 谱 这将允许比较有代表性的例子 在这两类酶中，尽管在其他方面无关， 同样的生物功能。