Crystallization of a Bacterial Single Stranded Annealing DNA Repair Protein

Abstract

INTRODUCTION: Single-stranded annealing (SSA) proteins bind to single-stranded DNA (ssDNA) and promote the pairing of homologous DNA strands, a process that is involved in the repair of double-stranded breaks. This work focuses on RecT and β protein, from Escherichia coli and bacteriophage lambda, respectively, which are two classic examples of SSA proteins. The human protein Rad52 promotes the same reactions as RecT and β, and is involved in multiple DNA repair pathways.

IMPORTANCE: Cells and DNA have mechanisms that repair mutations and failure to do so may lead to cancer. Chemotherapy requires study of repair mechanisms. Therefore, it is important to understand the DNA repair processes of RecT and β. Electron Microscopy studies propose that the proteins form oligomeric rings and helical filaments, but information on the fold of the proteins and the DNA binding sites on the surface of the oligomers is not available. This information can be obtained from high resolution images of a protein’s structure.

AIM: The aim of this study is to determine the mechanism by which SSA proteins bind to ssDNA and promote the annealing of complimentary DNA strands. This will increase our general understanding of how proteins recognize and repair damaged sites of DNA in cells. Our approach will employ the powerful technique of x-ray crystallography to determine the three-dimensional structure of SSA proteins at high resolution. This technique requires that the SSA protein be crystallized.

METHODS: A sample of RecT or β is purified and screened for initial crystals which are then optimized to grow larger crystals. Once large crystals form, x-ray diffraction is used to solve the three-dimensional atomic structure of the protein, preferably in complex with DNA substrates.

CONCLUSION: The first large crystals of RecT that can diffract to 6-7 Angstroms have been formed in the lab, which is a significant step toward understanding the protein repair mechanism, which will ultimately provide a foundation for improved applications in cancer treatment.