Structure and Mechanism of the Red beta Recombineering Enzyme

Ohio State University, The, Columbus OH

Investigators

Abstract

This project will study a process by which bacteria can be re-engineered for useful purposes such as conversion of carbon dioxide into biofuels to mitigate climate change, remediation of toxic waste, and killing cancer cells. Bacterial re-engineering involves DNA recombination whereby new DNA sequences are added to bacterial cells and incorporated into their genomes by a protein called recombinase. The recombinase binds to the input DNA and pairs it with the host bacterial genome for incorporation during replication. In order to work efficiently, the recombinase must interact with single-stranded DNA binding protein (SSB), which is part of the bacterial replication machinery. This project will study how the recombinase binds to SSB, by visualizing the complex at atomic resolution and by measuring the strength and speed of the interactions. New knowledge about the interactions, and the corresponding ability to manipulate them, will help increase the efficiency of DNA recombination and expand the utility of genome engineering in different types of bacteria for new applications. The project will also train future scientists, with particular emphasis on high school students. The study focuses on the Red-beta protein from bacteriophage lambda that is part of a DNA recombination system used for replication and repair of DNA breaks. Red-beta catalyzes single-stranded DNA annealing (SSA), and its activity has been harnessed for powerful methods of bacterial genome engineering. In these methods Red-beta binds to transformed synthetic DNA and anneals it to the existing bacterial genome as it is undergoing replication. This process requires interaction between Red-beta and SSB protein, which binds and protects the lagging strand of the replication fork. This project will use x-ray crystallography and cryo-electron microscopy to determine three-dimensional structures of the Red-beta-SSB complex, and other biophysical methods to determine the energetics of the binding interaction. The resulting knowledge will be used to fine-tune the Red-beta-SSB interaction and to make it compatible in a wider range of bacterial hosts. The project will also provide laboratory research training to students at all levels, with particular emphasis on high school students. This project is jointly funded by the Genetic Mechanisms and Molecular Biophysics programs of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.