Structure and mechanism of the red beta recombineering enzyme

这个项目的目标是了解细菌基因组工程强大的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前景，这些细菌被用作制造治疗和工业用化合物的工厂。这种名为Red beta的蛋白质与合成的DNA结合，并将其重组到细菌基因组中，进行所需的改变。值得注意的是，这种蛋白质可以同时修改基因组中的多个目标位置，使用每个位置的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推动细菌基因组的快速(但受控)进化。该项目的具体目标是绘制出接触和操纵DNA的蛋白质区域，了解蛋白质是如何工作的，并分离出工作更有效的蛋白质变体。希望的结果是开发出新的蛋白质，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。特别强调的是为附近一所高中的学生提供延长的、深入的培训机会，这所高中实施了一门以STEM为重点的创新课程。噬菌体lambda的β蛋白是红色重组系统的关键组成部分，该系统通过一种名为单链退火的机制促进DNA断裂的修复。由于它的效率和在相对较短的同源性区域工作的能力，该蛋白质已被用于细菌基因组工程的强大的新方法，称为重组工程和MAGE(多重自动化基因组工程)。然而，红色贝塔作用的分子机制却知之甚少。这个项目将通过以下方式增加我们对红色贝塔的理解：(1)使用化学足迹和质谱学来绘制出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；以及(3)进行遗传筛选，以分离出具有更高活性的蛋白质变体，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体中蛋白质与DNA的相对亲和力的变化如何影响其活体功能输出。该项目由生物科学局分子和细胞生物科学部的遗传机制小组和工程局化学、生物工程、环境和运输系统司的生物技术和生化工程计划共同资助。

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.