Structure and Specificity of Restriction-Modification (R-M) Systems

限制性修饰（R-M）系统的结构和特异性

• 批准号: 10727038
• 负责人: ANEEL K. AGGARWAL
• 金额: $ 9.51万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10727038
• 关键词: Archaea; Bacteria; Biotechnology; Communication; Complex; DNA; DNA Binding; DNA Restriction-Modification Enzymes; Distant; Engineering; Enzymes; Family; Genes; Innate Immune System; Knowledge; Learning; Medical Research; Medicine; Methylation; Modernization; Modification; Molds; Molecular; Mutate; Nobel Prize; Physiology; Proteins; Recombinant DNA; Site; Specificity; Structure; Technology; Time; endonuclease; helicase; insight; novel; nuclease; polypeptide; prevent; prototype; success; tool; viral DNA

Restriction-modification (R-M) systems comprise the innate immune system in bacteria and archaea. Their discovery ~50 years ago by Arber, Nathans, and Smith (1978 Nobel Prize in Physiology & Medicine) opened the doors of modern biotechnology. Without R-M enzymes there would haven been no recombinant DNA revolution and no gene technology, as we know it today. R-M systems range from simple Type II enzymes to more complex families of enzymes that require ATP (Type I and III) or that encode both endonuclease and methylation activities within the same polypeptide (Type IIL). Much has been learned over the past two decades about the structure and mechanism of the simple Type II enzymes (such as BamHI and FokI), providing fundamental insights into the basis of extreme protein-DNA selectivity and lending to the creation of novel chimeric nucleases. However, much remains to be learned about the other more complex families of R-M enzymes. EcoP15I is a prototype of the Type III R-M family that functions as a pseudo-helicase or a molecular switch to communicate between distant DNA sites. The DNA is cleaved when two EcoP15I complexes collide. Although Ecop15I was discovered >40 years ago there had been no structural information. We have resolved the crystal structure of the complete Ecop15I complex. We will carry out additional structural and functional studies aimed at understanding its mechanism of translocation and DNA cleavage. MmeI is a prototype of the Type IIL R-M family that provides a natural platform for engineering new DNA-binding specificities. Some success has already been achieved in this direction. We will use structural information on MmeI-like enzymes to identify specificity determinants, which can then be rationally mutated to generate new nucleases. We also look to understand how these enzymes control their nuclease activity, as a means to prevent self-restriction while at the same time allowing for restriction of viral DNA. Overall, we will uncover new structural principles by which these complex R-M systems communicate and cleave DNA over long distances and how specificity determinants can be molded to create new enzymes.

限制性修饰（R-M）系统包括细菌和古细菌的先天免疫系统。他们的 Discovery〜50年前，Arber，Nathans和Smith（1978年诺贝尔生理学与医学奖） 打开了现代生物技术的大门。没有R-M酶，就没有重组 众所周知，DNA革命，没有基因技术。 R-M系统范围从简单类型II 需要ATP（I型和III）或编码两者的更复杂酶家族的酶 核酸内切酶和甲基化活性（类型IIL）。已经学到了很多 在过去的二十年中，关于简单II型酶的结构和机制（例如 BAMHI和FOKI），为极端蛋白DNA选择性和 借给新型嵌合核酸酶的创造。但是，还有很多关于另一个 R-M酶的更复杂的家族。 Ecop15i是III型R-M家族的原型，该家族起作用 伪螺旋酶或分子开关，以在遥远的DNA位点进行通信。 DNA裂解 当两个Ecop15i复合物相撞时。尽管ecop15i被发现> 40年前没有 结构信息。我们已经解决了完整的Ecop15i复合物的晶体结构。我们将 进行其他旨在了解其易位机制的结构和功能研究 和DNA裂解。 MMEI是IIL R-M家族类型的原型，它为自然平台提供了 工程新的DNA结合特异性。在这个方向上已经取得了一些成功。我们 将使用有关MMEI样酶的结构信息来识别特异性决定因素，然后可以是 合理突变以产生新的核酸酶。我们还希望了解这些酶如何控制它们 核酸酶活性，作为预防自我限制的一种手段，同时允许病毒限制 脱氧核糖核酸。总体而言，我们将发现这些复杂的R-M系统进行通信的新结构原理 并在长距离上切割DNA，以及如何模制特异性决定因素以创建新的 酶。