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制和 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 裂解以启动 它们的内含子的移动性。 这些核酸内切酶的非凡之处在于 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 的副本。 该酶识别 14 以序列特异性方式的碱基对结合位点，切割靠近 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，可识别 35 个碱基对 DNA 以序列耐受方式靶向，切割 23 至 25 个核苷酸 内含子插入位点上游并留下 2-核苷酸 3'- 悬垂。 LAGLI-DADG 酶与其底物相互作用 主要通过DNA的大沟，I-TevI​​，最好 以 GIY-YIG 酶为特征，主要通过 小凹槽。 该项目的具体目标是确定三维 酶、分离域和复合物的结构 结合 X 射线晶体学和 NMR 来确定底物 光谱学。 这将允许比较代表性的例子 两类酶虽然在其他方面不相关，但携带 具有相同的生物学功能。