Doctor of Philosophy (PhD)
DNA protection during starvation (Dps) proteins are important for bacterial oxidative stress responses. This study aims to understand the roles and characteristics of the two Dps homologs in Deinococcus radiodurans: Dps-1 and Dps-2. Dps-2 contains a predicted signal peptide and in vivo localization of Dps-2 reveals that its location is non-cytoplasmic. β-galactosidase assays show that the Dps-2 promoter is upregulated in the presence of H2O2 and the results from the DNA protection assay show that Dps-2 is able to protect DNA efficiently against reactive oxygen species (ROS). Dps-2 has a C-terminal extension that is needed for assembly into a dodecamer but not for DNA binding. Dps-1 is assembled from six dimers and stoichiometric experiments reveal that the stoichiometry of Dps-1 binding to 22 bp DNA substrate is 1:6, meaning that Dps-1has six DNA binding sites. However, for the 26 bp DNA substrate, the stoichiometry is 1:4, suggesting that the protein can interact with both faces of a DNA duplex provided there are two consecutive major grooves on each face. Furthermore, mutation of the surface arginine (Arg132) causes a decrease in DNA binding, which indicates that this residue is involved in the path of DNA binding of Dps-1 after the initial contact is made with the N-terminus. A model for the mode of Dps-1 binding to genomic DNA is proposed based on these observations. Dps-1 has a unique metal site at the end of the N-terminal extension and mutations of this site cause the protein to exist as a hexamer and this lead to a significant reduction in DNA binding. The mutant protein (Dps-HE) breaks down into dimers and loses its ability to bind DNA upon removal of divalent metals, wherease removal of metals from full-length Dps-1 has no effect on oligomeric state. These findings suggest that the N-terminal metal site is needed for proper assembly, but once the protein oligomerizes to a dodecamer, metals are no longer required to maintain the dodecameric state. These results suggest that the role of Dps-1 might be to organize genomic DNA while the role of Dps-2 might be to provide protection against incoming ROS.
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Release the entire work immediately for access worldwide.
Nguyen, Khoa Huynh, "The roles of Deinococcus radiodurans Dps-1 and Dps-2 in nucleoid organization and in survival during oxidative stress" (2013). LSU Doctoral Dissertations. 2659.