Structure and mechanism of the red beta recombineering enzyme

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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.