Intron endonucleases and inteins

描述（由申请人提供）：归巢内切酶是一种罕见的DNA切割酶，通常由内含子和内含子编码。内含子内切酶和内含子内切酶因其分子机制、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备受关注。在过去的筹资期间，我们广泛合作，在实现上述四个具体目标方面取得了进展。首先，我们发现噬菌体T4组I内含子内切酶1-Tevl是GIY-YIG家族的一员，具有双重功能，可作为转录自抑制因子。其次，我们将古细菌l-Dmol内含子内切酶（单体LAGLIDADG家族酶）改造成异二聚体、同二聚体和特异性改变的单体，这一成就具有实践和进化意义。第三，我们通过低温电子显微镜（EM）第一次瞥见了乳球菌II组内含子编码的逆转录元件，该元件由具有核酸内切酶和逆转录酶（RT）活性的蛋白质与内含子RNA复合物组成，支架在核糖体上。最后，我们解决了分枝杆菌内蛋白的四个变体的结构，并深入了解了该内蛋白的切割机制。以这些结果为跳板，我们提出下一阶段研究的四个具体目标：探讨l-TevI连接体的结构和功能，该连接体作为催化和DNA结合域之间的沟通桥梁，控制裂解与抑制；为了验证连接子作为氧化还原开关的假设，它调节切割保真度，从而影响内含子的扩散。2. 利用冷冻电镜等技术，为II族内含子核糖核蛋白（RNP）的生化功能奠定结构基础，研究RNP与三大分子起始复合物中靶DNA的相互作用。3. 探讨果蝇Penelope逆转录因子，该因子结合了GIY-YIG内切酶和与端粒酶相关的RT，以确定它们之间的相互关系，特别是内切酶如何与端粒酶样RT通信以启动cDNA合成。4. 目的：研究分枝杆菌肠蛋白的自剪接和分散，提高肠蛋白抑制剂的有效性，使其既可作为机制探针，又可作为潜在的抗微生物药物。我们再次采取合作的、跨学科的方法，将遗传学、生物化学与计算生物学和结构生物学结合起来。通过这种方式，我们将提高我们对内切酶和内切酶的生物学和进化的理解，作为一种手段，提高它们作为生物技术试剂的有效性，并利用内切酶作为药物开发靶点的潜力。

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.