STRUCTURAL STUDIES OF INTRON ENCODED ENDONUCLEASES

这个应用程序的目标是研究作用机制和 两种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裂解启动 它们内含子的移动性。这些内切酶的显著之处在于 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它们的DNA底物。 I-DmoI和I-Tevi代表寻的的两个主要子类 核酸内切酶。I-DmoI是多数类的成员，包含两个 一个保守的主题的复制品，LAGLI-DADG。这种酶识别一种14 碱基对结合位点以序列特异的方式，裂解接近 内含子插入位点，并留下一个4-核苷酸3‘-突出。我- Tevi是GIY-YIG家族的成员，识别35个碱基对的DNA 以序列耐受的方式靶向，切割23到25个核苷酸 位于内含子插入位点的上游，并留下2-核苷酸3‘- 悬垂的。当LAGLI-DADG酶与底物相互作用时 主要是通过DNA的大沟，I-Tevi，最好的 以GIY-YIG酶为特征的，主要通过 小凹槽。 该项目的具体目标是确定三维 酶的结构、孤立结构域和与 X-射线结晶学和核磁共振相结合的基片 光谱学。这将允许比较有代表性的例子 两种酶中的一种，虽然在其他方面无关，但它们携带 具有相同的生物学功能。

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.