Studies On The Antioxidant Protein Dps (DRB0092) In Deinococcus Radiodurans

Deinococcus radiodurans is one of the most extraordinary radiation-resistant organisms on the earth. It also has a high resistance to desiccation, UV-ray, oxidative stress and a variety of DNA damaging agents. Although a lot of studies showed that these multiple resistant phenotypes was attributed to its anti-oxidant system and highly efficient DNA repair process, the mechanism of the extreme resistance in this bacterium is still unclear. Therefore, it is of significance to identify and study on the functional proteins which are involved in DNA repair and protection of D. radiodurans.Dps (DNA protection during starvation) is a member of iron-binding protein super-family which was found only in prokaryotes. Along with ferritin in eukaryotes and bacterioferritin, they constitute the superfamily of ferritin. Dps proteins have ferritin-like activity (iron storage-detoxification) and the ability to attenuate the production of ROS. Dps protein protects DNA by two mechanisms: 1) Dps proteins uptake and sequester ferrous iron so as to prevent the generation of highly reactive hydroxyl radicals through Fenton reaction induced by Fe²⁺ and H₂O₂; (2) Dps proteins provide a novel way of binding to DNA and tightly-packed, highly ordered crystalline of Dps-DNA complexes are formed. This physical sequestration limits the accessibility of DNA to detrimental factors such as free radical and DNase I.The genome of Deinococcus radiodurans embraces two genes that are predicted to belong to Dps family: gene DR2263 is encoded on chromosome I, and gene DRB0092 is on megaplasmid MP1. The research on biochemical property and crystal structure of DR2263 protein (Dps-1) was recently published. Our studies focus on gene function and protein property of DRB0092. The results were shown as below:1. Using the method of motif search in different database, we found that DRB0092 protein has a ferritin-like conserved domain, which indicated that DRB0092 protein is likely belonging to ferritin super-family. The multiple sequences alignment of DRB0092 protein with other Dps proteins using ClustalW software showed that protein sequence of DRB0092 did not have high identities with other Dps proteins. However, there are several lysines in N-terminal which were assumed to be involved in DNA binding. Other conserved residues such as His100,Asp127 and Glu131 were identified and assumed to constitute a conserved domain as ferroxidase center. Further analysis showed that 21aa (amino acids) chain existed in upstream of N-terminal was assumed to be a signal peptide in DRB0092 protein, which can not be found in other Dps proteins. This result indicated that DRB0092 protein may have some unique property and function.2. Based on the method of polymerase chain reaction in. vitro and homologous genetic recombination in vivo, the gene DRB0092 was deleted from the wide type strain R1 genome. Survival rates of the mutant and wild type strain were investigated after challenged with different concentration of ionizing radiation and hydrogen peroxide (H2O2). Results showed that the survival rate of dps mutant reduced rapidly compared to the wide type strain. However, the survival rate of the mutant was recovered after the DRB0092 gene was complemented into the mutant. To determinate the changes of catalase activity in dps mutant, an iron staining method was used after non-denatured polyacrylamide electrophoresis gels (Native-PAGE). The result displayed that two catalases in dps mutant were enhanced than that of wild type, especially under the challenge of hydrogen peroxide.3. The soluble DRB0092 protein was obtained by expression of recombinant protein in E. coli, and further biochemical experiments in vitro showed: Dps monomer assembled to trimers, tetramers and dodecamers in low salt buffer after being crosslinked with glutaraldehyde. Dps monomer could not interact with pUC19 plasmid, while polymeric assemblies could bind to plasmid DNA and provide efficient protection against hydroxyl radical-mediated DNA cleavage caused by Fe²⁺ and H₂O₂ through Fenton reaction.Based on these results, we conclude that Dps-2 protein (DRB0092) in D. radiodurans has a strong capacity of DNA binding and protection. It plays an important role in antioxidant system, which may contribute a great deal to the extreme resistance of D. radiodurans.