Intron endonucleases and inteins

内含子核酸内切酶和内含子

• 批准号: 6683666
• 负责人: MARLENE BELFORT
• 金额: $ 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6683666
• 关键词: Bacillus subtilis; DNA binding protein; Escherichia coli; Mycobacterium tuberculosis; SDS polyacrylamide gel electrophoresis; bacterial genetics; binding sites; biochemical evolution; cryoelectron microscopy; endonuclease; enzyme mechanism; enzyme structure; fluorescence resonance energy transfer; gene mutation; intein; introns; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; prokaryote; site directed mutagenesis

DESCRIPTION (provided by applicant): Homing endonucleases are rare-cutting enzymes that are most often encoded by introns and inteins. They function to cleave DNA and thereby initiate the homing reactions that mobilize their respective genetic elements. Homing enzymes are grouped into four families, based on conserved sequence elements, the LAGLIDADG, GIY-YIG, H-N-H and His-Cys motifs. Inteins are self-splicing proteins that are evolutionarily related to homing endonucleases. Interest in these enzymes is heightened by their phylogenetic diversity and the widespread occurrence of intron and intein homing, coupled with the unusual properties of the endonuclease-nucleic acid interactions. We have made considerable progress in understanding the structure, function and evolution of homing endonucleases and inteins during the past funding period. This work focused on the modularity of these enzymes, on their different modes of catalysis, and on the mechanism of intein action as well as the biotechnological utility of inteins. The proposed work will again combine genetic, biochemical and structural studies to achieve the overall goal of defining the molecular mechanism and evolution of homing endonucleases and inteins. We have selected two endonuclease families, encoded by either group I or group II introns, and an intein for further study. Our endonuclease choices are based on diversity of function of the enzymes, and whether they act independently or in concert with RNA. Our intein work is conducted on the RecA intein from the pathogen Mycobacterium tuberculosis. The four specific aims are as follows: 1. To extend our mechanistic appreciation of the modular GIY-YIG endonucleases, particularly I-TevI; 2. To understand the function of the disparate H-N-H endonuclease family, with I-TevIII and the ribonucleoprotein LtrA as examples; 3. To probe the structural evolution of homing endonucleases, and their acquisition of maturase function; 4. To gain further insight into intein function and to utilize inteins as novel drug targets. Thus, on one hand, our work will continue to expand the understanding of the molecular mechanism and evolution of the functionally and phylogenetically diverse endonucleases that initiate intron homing. On the other hand, the research will shed light on intein function, while exploring the potential of inteins in antimicrobial drug development.

描述（由申请人提供）：归巢核酸酶是稀有切割的酶，通常由内含子和内部核素编码。它们的功能可以裂解DNA，从而引发了动员其各自遗传因素的归巢反应。基于保守的序列元素，laglidg，giy-yig，h-h-h-h和his-cys主题，将归巢酶分为四个家族。 intein是与归巢核酸内切酶相关的自剪蛋白。这些酶的兴趣通过其系统发育多样性以及内含子和内丁归巢的广泛出现，再加上核酸内核酸酶核酸相互作用的异常特性而提高了人们的兴趣。在过去的资金期间，我们在理解归核心核酸内切核和内核的结构，功能和演变方面取得了长足的进步。这项工作的重点是这些酶的模块化，其不同的催化模式以及内素作用的机理以及内部的生物技术实用性。拟议的工作将再次结合遗传，生化和结构研究，以实现定义分子机制和归巢核酸内切蛋白和进化素的总体目标。我们选择了两个由I组或II组内含子编码的核酸内切酶家族，以及一个用于进一步研究的内核家族。我们的核酸内切酶的选择是基于酶功能的多样性，以及它们是独立起作用还是与RNA共同起作用。我们的内素工作是在病原体分枝杆菌结核病的Reca内进行的。四个具体目的如下：1。扩展我们对模块化giy-yig内切酶的机械欣赏，尤其是I-TEVI； 2。了解不同的H-H-H H-H内核酸酶家族的功能，并以I-Teviii和核糖核蛋白LTRA为例。 3。探测归巢核酸内切酶的结构演变及其对成熟酶功能的获取； 4。为了进一步了解内部元素功能，并利用内部素作为新的药物靶标。因此，一方面，我们的工作将继续扩展对启动内含子归巢的功能和系统发育多样性的核丝的分子机制和演变的理解。另一方面，该研究将阐明内部功能，同时探索抗微生物药物发育中的潜力。