ENDONUCLEASES OF INTERRUPTED PROKARYOTIC GENES

描述：贝尔福特博士发现了多个有趣的第一组 噬菌体T4中原核生物的内含子，并从那以后研究了它们的 剪接和移动的发生、结构和机制。这个 细菌内含子是自剪接的，尽管可能有 细胞中的辅因子，它们编码内切酶，影响它们的 通过特定的双链DNA断裂介导的基因的迁移率 转换事件。另一个新奇之处是，一些与进化相关的 核酸内切酶也是自剪接蛋白质(“内含子”)，它必须 精确地拼接以维持它们所居住的基因的编码区。 对这些课题的研究将在#年贝尔福博士的实验室中进行。 两项拨款之间的未来，一项是针对RNA剪接机制的，另一项是 运动中的DNA重组，以及内切酶的第二次重组 机制和进化。在以下方面取得了相当重要的进展 第一个领域，但具体目标的直接背景 本应用是对黄连蛋白的结构和作用机制的研究。 两种不同类型的内切酶，一种来自T4，另一种来自 古细菌。Belfort博士已经将领域结构描述为 这些酶及其DNA结合部位，并已暴露在一些 详细说明特定的DNA-蛋白质相互作用；例如，酶 T4的I-TEVI接触DNA的小沟，就像一个有限的 收集其他DNA结合蛋白。它的DNA结合部位有一些 不寻常的构象，它由单独的识别和 可能与蛋白质的结构域结构相匹配的切割区域。 贝尔福博士的实验室已经生产出了I-DmoI的晶体 (考古细菌)酶，承诺原子结构可能很快 存在，可能是酶-DNA复合体。他们研究了人类的进化 这些元素，例如发现内切酶的关联性 它们的常驻内含子和有机体不同，支持这一观点 内切酶和内含子是独立出现的。他们注意到 核酸内切酶靶标与自身周围环境的相似性， 这表明核酸内切酶在调节自身的移动性方面也起着同样的作用 整个内含子。 新拨款有两个主要目标。1)进一步了解 核酸内切酶的蛋白质-DNA相互作用：化学计量、结合 细节，DNA结合在活性中的作用，以及酶的结构域， 特别是保守的氨基酸基序的功能。他们会 对酶进行生化表征，并提供酶 用于原子结构测定。2)确定一个保守的 SUNY内含子中相邻的内切酶识别序列具有 调节功能，并试图从一个紧密的 相关的内切酶。

DESCRIPTION: Dr. Belfort discovered the multiply interesting group one introns of prokaryotes in phage T4, and has since studied their occurrence, structure, and mechanisms of splicing and mobility. The bacterial introns are self-splicing, although probably assisted by cofactors in the cell, and they encode endonucleases that effect their mobility through a specific double-strand DNA break-mediated gene conversion event. A further novelty is that some evolutionarily related endonucleases are also self-splicing proteins ("Inteins"), which must splice precisely to maintain the coding region of the genes they inhabit. Study of these subjects will be divided in Dr. Belfort's laboratory in the future between two grants, one for the mechanism of RNA splicing and DNA recombination in mobility, and the second one for endonuclease mechanism and evolution. There is considerable important progress in the first area, but the immediate background to the specific aims of this application is studies of the structure and mechanism of action of two different types of endonucleases, one from T4 and one from an archaebacterium. Dr. Belfort has characterized the domain structure of these enzymes and their DNA binding sites, and has exposed in some detail the specific DNA-protein interactions; for example, the enzyme I-TevI of T4 contacts the minor groove of DNA, like only a limited collection of other DNA-binding proteins. Its DNA binding site has some unusual conformation, and it consists of separate recognition and cleavage regions that may match the domain structure of the proteins. Dr. Belfort's laboratory has produced crystals of the I-DmoI (archaebacterial) enzyme, promising that atomic structures may soon exist, perhaps of enzyme-DNA complex. They have studied evolution of these elements, e.g. finding that the relatedness of the endonucleases and their resident introns and organisms differ, supporting the idea that the endonucleases and introns arose independently. They noted similarity of the targets of endonucleases to the own surrounds, suggesting that endonucleases mediate their own mobility as well as that of the whole intron. There are two main goals of the new grant. 1) To further understand the protein-DNA interactions of endonucleases: stoichiometry, binding details, role of DNA binding in activity, and domains of the enzymes, particularly the function of the conserved amino acid motifs. They will perform biochemical characterization of the enzymes, and provide enzyme for atomic structure determination. 2) To determine if a conserved adjacent endonuclease recognition sequence in the sunY intron has a regulatory function, and to attempt to evolve an intein from a closely related endonuclease.