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 约 50 年前由 Arber、Nathans 和 Smith（1978 年诺贝尔生理学与医学奖）发现的 打开了现代生物技术的大门。没有 R-M 酶就不会有重组 正如我们今天所知，DNA 革命并没有基因技术。 R-M 系统范围从简单的 II 型 酶到需要 ATP（I 型和 III 型）或编码两者的更复杂的酶家族 同一多肽（IIL 型）内的核酸内切酶和甲基化活性。学到了很多东西 在过去的二十年里，关于简单的 II 型酶（例如 BamHI 和 FokI），提供了对极端蛋白质-DNA 选择性基础的基本见解和 有助于创造新型嵌合核酸酶。然而，关于其他方面仍有很多需要了解的地方 更复杂的 R-M 酶家族。 EcoP15I 是 Type III R-M 系列的原型，其功能为 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 复合物碰撞时。尽管 Ecop15I 是在 40 多年前就被发现的，但当时还没有 结构信息。我们已经解析了完整 Ecop15I 复合物的晶体结构。我们将 进行额外的结构和功能研究，旨在了解其易位机制 和 DNA 切割。 MmeI 是 Type IIL R-M 系列的原型，为 设计新的 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 MmeI 样酶的结构信息来识别特异性决定簇，然后可以将其 合理突变产生新的核酸酶。我们还希望了解这些酶如何控制它们的 核酸酶活性，作为防止自我限制的手段，同时允许限制病毒 脱氧核糖核酸。总的来说，我们将揭示这些复杂的 R-M 系统通信的新结构原理 并长距离切割 DNA 以及如何塑造特异性决定簇以创造新的 酶。