Intron endonucleases and inteins

描述（由申请人提供）：归巢核酸内切酶是罕见的切割DNA切割酶，最常由内含子和内含肽编码。内含子核酸内切酶和内含肽因其分子机制、系统发育多样性、在基因组进化中的作用以及在研究、生物技术和医学中的应用而受到广泛关注。在过去的资助期间，我们广泛合作，在所有四个具体目标方面取得了进展。首先，我们表明噬菌体T4组I内含子核酸内切酶1-Tevl，GIY-YIG家族的成员，是双功能的，加倍作为转录自阻遏物。其次，我们将古细菌l-Dmol内含子核酸内切酶（一种单体LAGLIDADG家族酶）改造成异二聚体、同二聚体和具有改变的特异性的单体，这是一项具有实际意义和进化意义的成就。第三，我们第一次瞥见，通过冷冻电子显微镜（EM），乳球菌II组内含子编码的逆转录元件，由具有核酸内切酶和逆转录酶（RT）活性的蛋白质组成，与其内含子RNA复合，支架在核糖体上。最后，我们解决了分枝杆菌内含肽的四个变体的结构，并深入了解这种内含肽的切割机制。以这些结果为出发点，我们提出了以下四个具体的研究目标：1。为了探测的结构和功能的l-TevI连接器，作为催化和DNA结合域之间的通信桥梁，并控制切割与抑制;并测试的假设，连接器作为一个氧化还原开关，调节切割保真度，从而影响内含子的传播。2.利用cryo-EM等技术，为II组内含子核糖核蛋白（RNP）的生化功能奠定结构基础，并研究RNP与靶DNA在三大分子起始复合物中的相互作用。3.探讨果蝇Penelope逆转录元件，它结合了一个GIY-YIG内切酶与端粒酶相关的RT，以确定它们之间的相互关系，特别是如何内切酶与端粒酶样RT通信启动cDNA合成。4.目的研究结核杆菌内含肽的蛋白质自剪接和分散，提高内含肽抑制剂的作用效果，作为机制的探针和潜在的抗微生物药物。我们再一次采取合作的跨学科方法，将遗传学和生物化学与计算和结构生物学相结合。通过这种方式，我们将提高我们的核酸内切酶和内含肽的生物学和进化的理解，作为一种手段，以促进其作为生物技术试剂的有效性，并利用内含肽作为药物开发的目标的潜力。

DESCRIPTION (provided by applicant): Homing endonucleases are rare-cutting DNA cleavage enzymes that are most often encoded by introns and inteins. Intron endonucleases and inteins attract considerable attention for their molecular mechanisms, phylogenetic diversity, role in genome evolution, and application in research, biotechnology and medicine. In this past funding period, we collaborated broadly to make strides in all four previous specific aims. First, we showed that phage T4 group I intron endonuclease 1-Tevl, a member of the GIY-YIG family, is bifunctional, doubling as a transcriptional autorepressor. Second, we engineered the archaeal l-Dmol intron endonuclease, a monomeric LAGLIDADG family enzyme, into a heterodimer, a homodimer and a monomer with altered specificity, an accomplishment with both practical and evolutionary implication. Third, we caught a first glimpse, by cryo-electron microscopy (EM), of a lactococcal group II intron-encoded retroelement, consisting of the protein which has endonuclease and reverse transcriptase (RT) activity, in complex with its intron RNA, scaffolded on a ribosome. Finally, we solved the structure of four variants of a mycobacterial intein, and gained insight into this intein's cleavage mechanism. With these results as a springboard, we propose the following four specific aims for the next phase of research: 1. To probe the structure and function of the l-TevI linker, which serves as a communication bridge between catalytic and DNA binding domains and controls cleavage versus repression; and to test the hypothesis that the linker acts as a redox switch, that regulates cleavage fidelity and thereby influences intron spread. 2. To use cryo-EM, among other techniques, to lay the structural foundation for biochemical function of the group II intron ribonucleoprotein (RNP), and study RNP interactions with target DNA in the tri-macromolecular initiation complex for retromobility. 3. To explore the Drosophila Penelope retro-element, which combines a GIY-YIG endonuclease with an RT related to telomerase, to determine their interrelationship, and particularly how the endonuclease communicates with a telomerase-like RT to initiate cDNA synthesis. 4. To study protein self-splicing and dispersal of a mycobacterial intein, and improve the effectiveness of intein inhibitors, which serve both as probes of mechanism and as potential anti-microbial agents. Once again we are taking a collaborative, interdisciplinary approach, combining genetics and biochemistry with computational and structural biology. In this way, we will enhance our understanding of the biology and evolution of endonucleases and inteins, as a means to promote their effectiveness as biotechnological reagents and to exploit the potential of inteins as targets for drug development.