STRUCTURAL STUDIES OF INTRON ENCODED ENDONUCLEASES

内含子编码核酸内切酶的结构研究

• 批准号: 6181039
• 负责人: PATRICK M VAN ROEY
• 金额: $ 20.45万
• 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/6181039
• 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.

该应用的目的是研究作用机理和 两个内含子编码的内切酶的底物识别特性， i-dmoi和i-tevi，催化双链DNA裂解以启动 其内含子的流动性。 这些核酸内切酶非常出色 它们与冗长的识别序列相互作用。 他们拥有 精致的切割位点特异性并扭曲其DNA底物。 I-DMOI和I-TEVI代表了两个主要子类 核酸内切酶。 I-DMOI是多数级的成员，其中两个 保守的主题的副本，拉格里达。 这种酶识别14 碱基对结合位点以序列特异性的方式，裂解接近 内含子插入部位，并留下4-核苷酸3'-横向指标。 我- Tevi是GIY-YIG家族的成员，识别出35个碱基对DNA 靶向靶向的方式，裂解23至25个核苷酸 内含子插入部位的上游，并留下2-核苷酸3'-- 悬垂。 而拉格利辅助酶与底物相互作用 主要通过DNA的主要凹槽I-Tevi，最好的 以GIY-YIG酶为特征，主要通过 小凹槽。 该项目的具体目的是确定三维 酶的结构，孤立域和与复合物的结构 X射线晶体学和NMR结合的底物 光谱法。 这将允许比较代表性的例子 在两类酶中，尽管其他不相关，但 消除相同的生物学功能。