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.

限制性内切酶修饰(R-M)系统是细菌和古生物天然免疫系统的组成部分。他们的 约50年前Arber、Nathans和Smith的发现(1978年诺贝尔生理学和医学奖) 开启了现代生物技术的大门。没有R-M酶，就不会有重组 DNA革命和没有基因技术，正如我们今天所知的那样。R-M系统的范围从简单的类型II 酶到更复杂的需要ATP(类型I和类型III)或两者都编码的酶家族 同一多肽(III型)内的核酸内切酶和甲基化活性。我们学到了很多 在过去的二十年里，关于简单的II型酶的结构和机制(如 BamHI和FokI)，提供了对极端蛋白质-DNA选择性和 有助于产生新的嵌合核酸酶。然而，对于另一种情况，还有很多需要了解的地方 更复杂的R-M酶家族。EcoP15I是III型R-M家族的原型，其功能是 一种假解旋酶或分子开关，用于在遥远的DNA位点之间进行通信。DNA被切割 当两个EcoP15I复合体发生碰撞时。虽然生态文明40年前就被发现了，但至今还没有 结构信息。我们已经解析了完整的EcoP15I络合物的晶体结构。我们会 开展额外的结构和功能研究，以了解其易位机制 和DNA裂解。MMEI是IIL R-M系列的原型，它为 设计新的DNA结合特异体。在这个方向上已经取得了一些成功。我们 将使用MMEI样酶的结构信息来识别特异性决定因素，然后可以 合理突变以产生新的核酸酶。我们还希望了解这些酶是如何控制它们的 核酸酶活性，作为一种防止自我限制同时允许限制病毒的手段 DNA总体而言，我们将发现这些复杂的R-M系统进行通信所依据的新的结构原理 远距离切割DNA，以及如何塑造特异性决定因素来创造新的 酵素。