ENDONUCLEASES OF INTERRUPTED PROKARYOTIC GENES

中断的原核基因的核酸内切酶

• 批准号: 2901987
• 负责人: MARLENE BELFORT
• 金额: $ 34.41万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2901987
• 关键词: Archaea; DNA binding protein; Escherichia coli; Streptococcus lactis; bacterial genetics; bacteriophage T4; biochemical evolution; endonuclease; enzyme mechanism; enzyme structure; gene mutation; introns; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; prokaryote; site directed mutagenesis

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.

描述：贝尔福特博士发现了多有趣的第一组 内含子的原核生物在噬菌体T4，并研究了他们的 剪接和移动的发生、结构和机制。的 细菌内含子是自我剪接的，尽管可能有 细胞中的辅因子，它们编码核酸内切酶， 通过特异性双链DNA断裂介导基因的迁移率 转换事件。另一个新奇之处在于， 核酸内切酶也是自剪接蛋白（“内含肽”），其必须 精确地拼接以维持它们所在基因的编码区。 这些受试者的研究将于年在贝尔福特博士的实验室进行。 两个赠款之间的未来，一个是RNA剪接的机制， 第二种是核酸内切酶的重组 机制与演化在以下方面取得了重大进展： 第一个领域，但具体目标的直接背景， 本申请是研究的结构和作用机制， 两种不同类型的核酸内切酶，一种来自T4，一种来自 古细菌贝尔福特博士已经描述了 这些酶及其DNA结合位点，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与DNA的小沟接触，就像只有一个有限的 其他DNA结合蛋白的集合。它的DNA结合位点有一些 不寻常的构象，它包括单独的识别和 可能与蛋白质的结构域结构相匹配的切割区域。 博士贝尔福特的实验室已经生产出了I-DmoI晶体 （古细菌）酶，有望原子结构可能很快 可能是酶-DNA复合物。他们研究了 这些元素，例如，发现核酸内切酶的相关性 它们的固有内含子和有机体也不同， 核酸内切酶和内含子是独立产生的。他们指出 核酸内切酶的靶与其自身周围的相似性， 这表明核酸内切酶介导它们自身的迁移性， 整个intron。 新赠款有两个主要目标。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， 特别是保守氨基酸基序的功能。他们将 进行酶的生化表征，并提供酶 用于原子结构测定。2)为了确定是否保守的 sunY内含子中的相邻核酸内切酶识别序列具有 调节功能，并试图从一个紧密的 相关的核酸内切酶。