Intron endonucleases and inteins

描述（由申请人提供）：归巢内切酶是稀有切割酶，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，调动各自的遗传元素。根据保守序列元件LAGLIDADG、GIY-YIG、H-N-H和His-Cys基序，归巢酶可分为4个家族。内部蛋白是自剪接蛋白，在进化上与归巢内切酶相关。这些酶的系统发育多样性、内含子和内含蛋白归巢的广泛存在，以及内切酶-核酸相互作用的不寻常特性，提高了人们对它们的兴趣。在过去的资助期内，我们在了解归巢内切酶和内含物的结构、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，它们的不同的催化模式，以及相互作用的机制以及相互作用的生物技术用途。这项工作将再次结合遗传学、生物化学和结构研究，以实现确定归巢内切酶和内含物的分子机制和进化的总体目标。我们选择了两个由I族或II族内含子编码的内切酶家族和一个内切酶进行进一步的研究。我们选择的内切酶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。我们的内部工作是对来自病原体结核分枝杆菌的RecA内部进行的。具体目标如下：1。扩展我们对模块化GIY-YIG内切酶，特别是I-TevI的机制评价；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. 探讨归巢内切酶的结构演化及其成熟酶功能的获得；4. 进一步了解肠内蛋白的功能，利用肠内蛋白作为新的药物靶点。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内切酶的分子机制和进化的理解。另一方面，该研究将揭示肠蛋白的功能，同时探索肠蛋白在抗菌药物开发中的潜力。

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.