Functional comparison of Deinococcus radiodurans Dps proteins suggests distinct in vivo roles.

Deinococcus radiodurans exhibits extreme resistance to DNA damage and is one of only few bacteria that encode two Dps (DNA protection during starvation) proteins. Dps-1 was shown previously to bind DNA with high affinity and to localize to the D. radiodurans nucleoid. A unique feature of Dps-2 is its predicted signal peptide. In the present paper, we report that Dps-2 assembly into a dodecamer requires the C-terminal extension and, whereas Dps-2 binds DNA with low affinity, it protects against degradation by reactive oxygen species. Consistent with a role for Dps-2 in oxidative stress responses, the Dps-2 promoter is up-regulated by oxidative stress, whereas the Dps-1 promoter is not. Although DAPI (4',6-diamidino-2-phenylindole) staining of Escherichia coli nucleoids shows that Dps-1 can compact genomic DNA, such nucleoid condensation is absent from cells expressing Dps-2. A fusion of EGFP (enhanced green fluorescent protein) to the Dps-2 signal peptide results in green fluorescence at the perimeter of D. radiodurans cells. The differential response of the Dps-1 and Dps-2 promoters to oxidative stress, the distinct cellular localization of the proteins and the differential ability of Dps-1 and Dps-2 to attenuate hydroxyl radical production suggest distinct functional roles; whereas Dps-1 may function in DNA metabolism, Dps-2 may protect against exogenously derived reactive oxygen species.

On the stoichiometry of Deinococcus radiodurans Dps-1 binding to duplex DNA.

DNA protection during starvation (Dps) proteins, dodecameric assemblies of four-helix bundle subunits, contribute to protection against reactive oxygen species. Deinococcus radiodurans, which is characterized by resistance to DNA damaging agents, encodes two Dps homologs, of which Dps-1 binds DNA with high affinity. DNA binding requires N-terminal extensions preceding the four-helix bundle core. Composed of six Dps-1 dimers, each capable of DNA binding by N-terminal extensions interacting in consecutive DNA major grooves, dodecameric Dps-1 would be predicted to feature six DNA binding sites. Using electrophoretic mobility shift assays and intrinsic tryptophan fluorescence, we show that dodecameric Dps-1 binds 22-bp DNA with a stoichiometry of 1:6, consistent with the existence of six DNA binding sites. The stoichiometry of Dps-1 binding to 26-bp DNA is 1:4, suggesting that two Dps-1 dodecamers can simultaneously occupy opposite faces of this DNA. Mutagenesis of an arginine (Arg132) on the surface of Dps-1 leads to a reduction in DNA binding. Altogether, our data suggest that duplex DNA lies along the dimer interface, interacting with Arg132 and the N-terminal α-helices, and they extend the hexagonal packing model for Dps-DNA assemblies by specifying the basis for occupancy of available DNA binding sites.

Determining the role of metal binding in protein cage assembly.

Assembly of protein cages may require structural metal ions to nucleate or stabilize association of protein subunits. We describe here an approach to establishing the role of metal ions in protein cage assembly and stability, focusing on detecting the presence of structural metal ions as well as establishing oligomeric state. A colorimetric assay for detection of metal is described, along with a gel filtration assay to assess protein cage assembly and a fluorescence-based method for determining protein stability.