Structure and mechanism of the red beta recombineering enzyme

红β重组酶的结构和机制

• 批准号: 1616105
• 负责人: Charles Bell
• 金额: $ 61.48万
• 依托单位: Ohio State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616105&HistoricalAwards=false
• 关键词: Structure; mechanism; red; beta; recombineering

The goal of this project is to understand the mechanism of the protein behind powerful new methods for bacterial genome engineering. These methods show promise for developing new strains of bacteria for use as factories for making compounds for therapeutic and industrial purposes. The protein, called red beta, binds to synthetic DNA and recombines it into the bacterial genome to make desired alterations. Remarkably, the protein can modify multiple target sites in the genome simultaneously, using libraries of DNA molecules for each site, while selecting for a desired functional output. Thus, the protein can drive rapid (but controlled) evolution of bacterial genomes in the laboratory. The specific goals of the project are to map out the regions of the protein that contact and manipulate the DNA, to understand how the protein works, and to isolate variants of the protein that work more efficiently. The desired outcome is to develop new proteins that can be used to improve and expand the current methods for bacterial genome engineering. The project will also provide training for graduate, undergraduate, and high school students. A particular emphasis will be to provide extended, in-depth training opportunities for students from a nearby high school that implements an innovative, STEM-focused curriculum.The beta protein of bacteriophage lambda is a key component of the red recombination system that promotes the repair of DNA breaks by a mechanism called single-strand annealing. Due to its efficiency and its ability to work at relatively short regions of homology, the protein has been exploited in powerful new methods for bacterial genome engineering known as recombineering and MAGE (Multiplex Automated Genome Engineering). However, the molecular mechanism by which red beta operates is poorly understood. This project will increase our understanding of red beta by (1) using chemical footprinting and mass spectrometry to map out specific residues of the protein that contact the DNA in the different complexes that are relevant to reaction, (2) performing mutational analyses to determine the importance of these residues in DNA binding and single strand annealing in vivo, and (3) performing a genetic screen to isolate variants of the protein with increased activity for single strand annealing in vivo. A particular emphasis is to understand how changes in the relative affinity of the protein for DNA in the different complexes that are relevant to the reaction impact its functional output in vivo.This project is funded jointly by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences, Directorate for Biological Sciences, and the Biotechnology and Biochemical Engineering Program in the Division of Chemical, Bioengineering, Environmental and Transport Systems, Directorate for Engineering.

该项目的目标是了解细菌基因组工程强大新方法背后的蛋白质机制。这些方法显示出开发新的细菌菌株的希望，这些菌株可以作为生产用于治疗和工业用途的化合物的工厂。这种被称为红β的蛋白质与合成DNA结合，并将其重组到细菌基因组中，以做出所需的改变。值得注意的是，该蛋白可以同时修改基因组中的多个目标位点，使用每个位点的DNA分子文库，同时选择所需的功能输出。因此，这种蛋白质可以在实验室中驱动细菌基因组的快速（但受控的）进化。该项目的具体目标是绘制出蛋白质接触和操纵DNA的区域，了解蛋白质如何工作，并分离出更有效地工作的蛋白质变体。期望的结果是开发新的蛋白质，可以用来改进和扩展目前的细菌基因组工程方法。该项目还将为研究生、本科生和高中生提供培训。一个特别的重点将是为附近一所高中的学生提供扩展的、深入的培训机会，该高中实施了创新的、以stem为重点的课程。噬菌体的β蛋白是红色重组系统的关键组成部分，该系统通过一种称为单链退火的机制促进DNA断裂的修复。由于其效率和在相对较短的同源区域工作的能力，该蛋白已被用于细菌基因组工程的强大新方法，即重组和MAGE（多重自动基因组工程）。然而，人们对红β的分子机制知之甚少。该项目将通过(1)使用化学足迹和质谱法来绘制与反应相关的不同复合物中与DNA接触的蛋白质的特定残基，(2)进行突变分析以确定这些残基在DNA结合和体内单链退火中的重要性，从而增加我们对红色β的理解。(3)进行遗传筛选，以分离具有增加活性的蛋白质变体，以便在体内进行单链退火。特别强调的是了解与反应相关的不同复合物中蛋白质对DNA的相对亲和力的变化如何影响其在体内的功能输出。该项目由生物科学理事会分子和细胞生物科学部的遗传机制组和工程理事会化学、生物工程、环境和运输系统部的生物技术和生化工程项目共同资助。