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.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-03042013-205101 |
| Date | 07 March 2013 |
| Creators | Nguyen, Khoa Huynh |
| Contributors | Grove, Anne, Doerrler, William, Ding, Huangen, Lee, Yong-Hwan, Eades, Susan |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-03042013-205101/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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