| This thesis is a biochemical study focused on understanding the regulation of the RecA protein in the bacteria Deinococcus radiodurans and Escherichia coli. D. radiodurans is a bacterium that can withstand up to 15,000 Gy of ionizing radiation (IR). The recA gene in D. radiodurans is essential for this radiation resistance. In Chapter 2, I will describe how the DrRecA protein is different from eubacterial proteins. DrRecA can bind to double-stranded DNA (dsDNA), or to single-stranded DNA (ssDNA) in the presence of the single-stranded DNA binding protein (SSB), without hydrolyzing ATP. ATP hydrolysis by the DrRecA-dsDNA filament can be stimulated with the addition of ssDNA, with or without DrSSB. In addition, the DrSSB protein can trigger a transfer of DrRecA molecules from the dsDNA to ssDNA. DrSSB is also essential for initiating and promoting homologous DNA strand exchange by DrRecA. I have also characterized the DrRecA G82S variant that has lower DNA binding ability and lower ATPase activity than WT DrRecA (Appendix A). ATPase activity of the DrRecA G82S-dsDNA filament cannot be activated by the addition of ssDNA, with or without DrSSB, contrary to the WT DrRecA protein. Additionally, the DrRecA G82S variant is recombinase dead.;A collaborate with Jim Keck's lab at UW-Madison has led to a structural and an in vivo characterization of the D. radiodurans SSB protein, which I will describe in Chapter 3. We have solved the structure of the DrSSB homodimer complex with ssDNA, and have shown by electron microscopy that DrSSB can form distinct complexes with DNA that is salt dependent, similarly to EcSSB. The DrSSB protein level in the cell increases post IR, and its localization is highly dynamic, forming dispersed puncta in the nucleoid immediately after IR treatment, followed by a single concentrated focus in the nucleoid at one hour of recovery post IR.;In Chapter 4, I will describe the loading mechanism of the E. coli RecA protein onto SSB-coated ssDNA by the RecOR proteins, also from E. coli. In collaboration with Sergey Korolev's lab at Saint Louis University, we used the EcRecR K23A/R27A (RecR KARA) or K23E/R27E (RecR KERE) variants that bind to EcRecO, but not EcRecF, as a tool to decipher the important steps in RecA loading. We discovered that the EcRecO-SSB and EcRecO-DNA interactions are crucial for EcRecA loading, and that EcRecA may be recruited by a direct protein-protein interaction with the EcRecR protein in solution. |
