The N-terminal Extensions of Deinococcus radiodurans Dps-1 Mediate DNA Major Groove Interactions as well as Assembly of the Dodecamer

Abstract

Dps (DNA protection during starvation) proteins play an important role in the protection of prokaryotic macromolecules from damage by reactive oxygen species. Previous studies have suggested that the lysine-rich N-terminal tail of Dps proteins participates in DNA binding. In comparison with other Dps proteins, Dps-1 from Deinococcus radiodurans has an extended N terminus comprising 55 amino acids preceding the first helix of the 4-helix bundle monomer. In the crystal structure of Dps-1, the first ϳ30 N-terminal residues are invisible, and the remaining 25 residues form a loop that harbors a novel metalbinding site. We show here that deletion of the flexible N-terminal tail obliterates DNA/Dps-1 interaction. Surprisingly, deletion of the entire N terminus also abolishes dodecameric assembly of the protein. Retention of the N-terminal metal site is necessary for formation of the dodecamer, and metal binding at this site facilitates oligomerization of the protein. Electrophoretic mobility shift assays using DNA modified with specific major/minor groove reagents further show that Dps-1 interacts through the DNA major groove. DNA cyclization assays suggest that dodecameric Dps-1 does not wrap DNA about itself. A significant decrease in DNA binding affinity accompanies a reduction in duplex length from 22 to 18 bp, but only for dodecameric Dps-1. Our data further suggest that high affinity DNA binding depends on occupancy of the N-terminal metal site. Taken together, the mode of DNA interaction by dodecameric Dps-1 suggests interaction of two metal-anchored N-terminal tails in successive DNA major grooves, leading to DNA compaction by formation of stacked protein-DNA layers.All aerobic microorganisms are exposed to reactive oxygen species ( OH ϩ Fe 3ϩ ) (1). Thus, the presence of iron increases the probability of oxidative damage to cellular components. Dps, initially studied in Escherichia coli, was shown to protect DNA by its ability to chelate ferrous iron and also by its physical association with DNA (2-5).Twelve Dps monomers form a spherical assembly similar to the spherical shell formed by 24 subunits of the iron storage protein, ferritin (6 -9). Each Dps monomer adopts a four-helix (A-D) bundle conformation as seen for ferritin, but unlike ferritin, Dps possesses a short helix in the middle of the BC loop and lacks the C-terminal fifth helix present in the ferritin monomer (10 -13). Secondly, the ferroxidase site in Dps is usually generated at the interface between two subunits and, with one reported exception, is not within the four-helix bundle as in the case of ferritin (14,15). It is also notable that not all Dps homologs follow the same catalytic mechanism, as exemplified by the absence of a conserved ferroxidase center in Lactococcus lactis DpsB and the failure of Bacillus anthracis Dps1 to utilize H 2 O 2 in the ferroxidation reaction (16,17).In contrast to the highly conserved ferroxidase center, Dps homologs have a variable N-terminal extension. This N-terminal tail, which contains multiple positively ...

Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

“…S3 in the supplemental material), suggesting assembly of a dodecameric particle like that expected for DPS or DPSL, as opposed to the 24-mer common to ferritin and bacterioferritin. While members of the ferritin superfamily are often found as stable oligomers, their assembly states can also be dependent upon changes in pH, salt concentration, metal ions, and temperature (8,12,14,20,30). However, despite surveying a variety of protein concentrations and buffer conditions, we were unable to identify conditions that give a stable dodecamer on the SEC column.…”

“…Assays were performed as described previously (8) moles of subunit) in 0.1 M bis-Tris, pH 6.5, 0.3 M NaCl at 22°C. E. coli DPS was used as a positive control, and bovine serum albumin (BSA) was used as a negative control.…”

Oxidative Stress Resistance inDeinococcus radiodurans

“…HU binds to four-way DNA junctions and may be additionally optimized for DNA recombination events by stabilizing recombination intermediates (208,209). Contrary to HU, one of the D. radiodurans Dps ("DNA protection during starvation") proteins, Dps1 (DR2263), was shown to promote DNA compaction in vitro (54) but not in vivo, as its depletion has no effect on genome condensation and cell viability (459). The D. radiodurans SMC ("structural maintenance of chromosomes") protein (DR1471), which is important for genome stabilization and DNA repair in eukaryotes (100, 365) and bacteria (640), has no role in genome condensation in D. radiodurans either (140a).…”

“…Editing of the pairing process by mismatch repair proteins is also expected to forestall recombination between nonhomologous fragments (373,490,510). Finally, tethering the ends of a given DSB to prevent them from dissociating via the cohesin-like RecN protein (518) or via genome condensation-promoting proteins, such as HU (459) and Dps1 (54) …”

SEp22, Salmonella Dps, a Key Molecule Bearing Both Pathogenicity and Resistance to Environmental Stresses in Salmonella

“…Intensive structural research, especially using with X-ray crystallography, has revealed a highly conserved structural fold of proteins within the Dps protein family isolated from different organisms. 12,16,17,[19][20][21][22][23][24][25] However, most of these studies mainly showed structural features common to other ferritins characterized in bacteria, including the paradigmatic ferritin (FtnA) and the hemecontaining bacterioferritin (Bfr), which are composed of at least 12 subunits folded into four-helix bundles and assembled into spherical protein shells as Dps is. 13,26) E. coli Dps has a very compact and stable structure with protrusion of the highly flexible and lysine-rich N-terminus (Fig.…”