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(申请人提供)：归巢内切酶是一种稀有的切割酶，通常由内含子和内含子编码。它们的功能是切割DNA，从而启动归巢反应，从而调动各自的遗传要素。根据保守的序列元件，归巢酶分为四个家族：LAGLIDADG、GIY-YIG、H-N-H和His-Cys基序。内含子是一种自我剪接的蛋白质，在进化上与归巢内切酶相关。这些酶的系统发育多样性和广泛存在的内含子和内含子归位，加上核酸内切酶-核酸相互作用的不同寻常的性质，增加了人们对这些酶的兴趣。在过去的资助期间，我们在理解归巢核酸内切酶和内含子的结构、功能和进化方面取得了相当大的进展。这项工作的重点是这些酶的模块化，它们不同的催化模式，内含素的作用机制以及内含素的生物技术用途。拟议的工作将再次结合遗传、生化和结构研究，以实现定义归巢核酸内切酶和内含子的分子机制和进化的总体目标。我们选择了两个内切酶家族，分别由第一或第二组内含子编码，以及一个内含子用于进一步研究。我们的核酸内切酶选择是基于酶功能的多样性，以及它们是独立作用还是与RNA协同作用。我们的内含素工作是在来自结核分枝杆菌的RecA内含子上进行的。四个具体目标如下：1.扩大我们对模块化GIY-YIG内切酶，特别是I-TEVI的认识；2.了解不同的H-N-H内切酶家族的功能，以I-TevIII和核糖核蛋白LTRA为例；3.探索归巢内切酶的结构演变及其成熟酶功能的获得；4.进一步了解内含子的功能，并利用内含子作为新的药物靶点。因此，一方面，我们的工作将继续扩大对启动内含子归巢的功能和系统发育多样性的内切酶的分子机制和进化的理解。另一方面，这项研究将揭示内含子的功能，同时探索内含子在抗菌药物开发中的潜力。