The N-terminal Extensions of Deinococcus radiodurans Dps-1 Mediate DNA Major Groove Interactions as well as Assembly of the Dodecamer
2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês
10.1074/jbc.m611255200
ISSN1083-351X
AutoresGargi Bhattacharyya, Anne Grove,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoDps (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 ∼30 N-terminal residues are invisible, and the remaining 25 residues form a loop that harbors a novel metal-binding 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. 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 ∼30 N-terminal residues are invisible, and the remaining 25 residues form a loop that harbors a novel metal-binding 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 (ROS) 2The abbreviations used are: ROS, reactive oxygen species; Dps, DNA protection during starvation; Dps-dn, Dps-1 deleted for its N-terminal extension; Dps-met, N-terminally truncated Dps-1 retaining the metal site; TBP, TATA box-binding protein; FPLC, fast protein liquid chromatography; Mops, 4-morpholinepropanesulfonic acid; BSA, bovine serum albumin; DMS, dimethyl sulfate; TBE, Tris borate-EDTA. such as O2.¯, and ·OH that can damage cellular macromolecules, including proteins, lipids, and DNA. Therefore, they have developed a number of defense mechanisms to combat such stress conditions in the environment. One of the key components in the response to oxidative stress in prokaryotes is the nonspecific DNA-binding protein Dps (DNA protection during starvation). Toxicity of the ROS H2O2 itself is relatively weak, but it can form highly reactive hydroxyl radicals in the presence of transition metals such as Fe2+ according to the Fenton reaction (H2O2 + Fe2+ →·OH + -OH + Fe3+) (1Crichton R.R. Wilmet S. Legssyer R. Ward R.J. J. Inorg. Biochem. 2002; 91: 9-18Crossref PubMed Scopus (404) Google Scholar). 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 (2Almiron M. Link A.J. Furlong D. Kolter R. Genes Dev. 1992; 6: 2646-2654Crossref PubMed Scopus (625) Google Scholar, 3Ilari A. Ceci P. Ferrari D. Rossi G.L. Chiancone E. J. Biol. Chem. 2002; 277: 37619-37623Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 4Martinez A. Kolter R. J. Bacteriol. 1997; 179: 5188-5194Crossref PubMed Google Scholar, 5Zhao G. Ceci P. Ilari A. Giangiacomo L. Laue T.M. Chiancone E. Chasteen N.D. J. Biol. Chem. 2002; 277: 27689-27696Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Twelve Dps monomers form a spherical assembly similar to the spherical shell formed by 24 subunits of the iron storage protein, ferritin (6Boyd D. Vecoli C. Belcher D.M. Jain S.K. Drysdale J.W. J. Biol. Chem. 1985; 260: 11755-11761Abstract Full Text PDF PubMed Google Scholar, 7Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2279) Google Scholar, 8Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-448Crossref PubMed Scopus (281) Google Scholar, 9Theil E.C. Matzapetakis M. Liu X. J. Biol. Inorg. Chem. 2006; 11: 803-810Crossref PubMed Scopus (154) Google Scholar). 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 (10Bozzi M. Mignogna G. Stefanini S. Barra D. Longhi C. Valenti P. Chiancone E. J. Biol. Chem. 1997; 272: 3259-3265Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 11Grant R.A. Filman D.J. Finkel S.E. Kolter R. Hogle J.M. Nat. Struct. Biol. 1998; 5: 294-303Crossref PubMed Scopus (445) Google Scholar, 12Papinutto E. Dundon W.G. Pitulis N. Battistutta R. Montecucco C. Zanotti G. J. Biol. Chem. 2002; 277: 15093-15098Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 13Zanotti G. Papinutto E. Dundon W. Battistutta R. Seveso M. Giudice G. Rappuoli R. Montecucco C. J. Mol. Biol. 2002; 323: 125-130Crossref PubMed Scopus (128) Google Scholar). 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 (14Ilari A. Stefanini S. Chiancone E. Tsernoglou D. Nat. Struct. Biol. 2000; 7: 38-43Crossref PubMed Scopus (229) Google Scholar, 15Gauss G.H. Benas P. Wiedenheft B. Young M. Douglas T. Lawrence C.M. Biochemistry. 2006; 45: 10815-10827Crossref PubMed Scopus (52) Google Scholar). 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 H2O2 in the ferroxidation reaction (16Stillman T.J. Upadhyay M. Norte V.A. Sedelnikova S.E. Carradus M. Tzokov S. Bullough P.A. Shearman C.A. Gasson M.J. Williams C.H. Artymiuk P.J. Green J. Mol. Microbiol. 2005; 57: 1101-1112Crossref PubMed Scopus (56) Google Scholar, 17Liu X. Kim K. Leighton T. Theil E.C. J. Biol. Chem. 2006; 281: 27827-27835Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In contrast to the highly conserved ferroxidase center, Dps homologs have a variable N-terminal extension. This N-terminal tail, which contains multiple positively charged residues, extends from the four-helix bundle core into the solvent (11Grant R.A. Filman D.J. Finkel S.E. Kolter R. Hogle J.M. Nat. Struct. Biol. 1998; 5: 294-303Crossref PubMed Scopus (445) Google Scholar, 18Kim S.G. Bhattacharyya G. Grove A. Lee Y.H. J. Mol. Biol. 2006; 361: 105-114Crossref PubMed Scopus (39) Google Scholar, 19Romao C.V. Mitchell E.P. McSweeney S. J. Biol. Inorg. Chem. 2006; 11: 891-902Crossref PubMed Scopus (46) Google Scholar). Because the surface of the Dps protein does not display "classical" DNA binding motifs and is dominated by negative charges, it has been proposed that the DNA binding properties of E. coli Dps, the family prototype, are associated with the presence of the lysine-rich N-terminal tail (11Grant R.A. Filman D.J. Finkel S.E. Kolter R. Hogle J.M. Nat. Struct. Biol. 1998; 5: 294-303Crossref PubMed Scopus (445) Google Scholar). Consistent with this notion, proteins that do not have an N-terminal extension such as the Dps homolog Hp-NAP from Helicobacter pylori or Dps from Agrobacterium tumefaciens, whose N-terminal tail is immobilized on the protein surface, fail to bind DNA (20Ceci P. Ilari A. Falvo E. Chiancone E. J. Biol. Chem. 2003; 278: 20319-20326Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 21Tonello F. Dundon W.G. Satin B. Molinari M. Tognon G. Grandi G. Del Giudice G. Rappuoli R. Montecucco C. Mol. Microbiol. 1999; 34: 238-246Crossref PubMed Scopus (147) Google Scholar). The mesophilic, non-spore-forming eubacterium Deinococcus radiodurans is known for its extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents, such as ionizing radiation and desiccation, both conditions that are characterized by the presence of oxidative radicals (22Battista J.R. Annu. Rev. Microbiol. 1997; 51: 203-224Crossref PubMed Scopus (428) Google Scholar, 23Wilson III, D.M. Sofinowski T.M. McNeill D.R. Front. Biosci. 2003; 8: 963-981Crossref PubMed Google Scholar). D. radiodurans encodes two proteins that are predicted to belong to the Dps family of proteins. The Dps homolog most closely related to E. coli Dps (Dps-1, the product of locus DR2263) is encoded on chromosome 1. Recently it was shown that both dodecameric and dimeric forms of Dps-1 can bind DNA and that both exhibit ferroxidase activity (24Grove A. Wilkinson S.P. J. Mol. Biol. 2005; 347: 495-508Crossref PubMed Scopus (54) Google Scholar). Notably, the dodecameric Dps-1 is functionally distinct from other Dps homologs because of its inability to provide efficient protection against hydroxyl radical-mediated DNA degradation. The N-terminal extension of Dps-1 is longer than that of E. coli Dps and contains a total of seven lysine residues. The recently solved crystal structure of D. radiodurans Dps-1 shows that the N terminus is exposed at the surface of the dodecamer and would be available to interact with DNA (18Kim S.G. Bhattacharyya G. Grove A. Lee Y.H. J. Mol. Biol. 2006; 361: 105-114Crossref PubMed Scopus (39) Google Scholar, 19Romao C.V. Mitchell E.P. McSweeney S. J. Biol. Inorg. Chem. 2006; 11: 891-902Crossref PubMed Scopus (46) Google Scholar). The first ∼30 amino acids of the N terminus are not visible in the structure and are presumably disordered. The crystal structure of Dps-1 also reveals a unique metal-binding site located at the base of the N-terminal tail, docking it to the outer surface of the protein. Only L. lactis Dps features a metal site at the N terminus; however, there is no structural similarity between L. lactis Dps and D. radiodurans Dps-1 in this region (16Stillman T.J. Upadhyay M. Norte V.A. Sedelnikova S.E. Carradus M. Tzokov S. Bullough P.A. Shearman C.A. Gasson M.J. Williams C.H. Artymiuk P.J. Green J. Mol. Microbiol. 2005; 57: 1101-1112Crossref PubMed Scopus (56) Google Scholar, 18Kim S.G. Bhattacharyya G. Grove A. Lee Y.H. J. Mol. Biol. 2006; 361: 105-114Crossref PubMed Scopus (39) Google Scholar). Here we show that the N-terminal extension of Dps-1 surprisingly is required not only for DNA binding but also for assembly of the dodecamer. Analysis of DNA binding suggests a mode of interaction consistent with metal-anchored N-terminal extensions interacting in successive DNA major grooves. With multiple DNA binding sites, this mode of interaction is consistent with the previously observed DNA compaction, suggested to arise as a consequence of the formation of stacked layers of DNA and protein (25Frenkiel-Krispin D. Ben-Avraham I. Englander J. Shimoni E. Wolf S.G. Minsky A. Mol. Microbiol. 2004; 51: 395-405Crossref PubMed Scopus (110) Google Scholar). Mutagenesis, Overexpression, and Purification of Dps-dn and Dps-met—Deletion of the entire N-terminal extension (Dps-dn) was accomplished by amplifying the Dps-1 gene lacking 165 bp at the N terminus from plasmid containing the entire Dps-1 gene (pET5a-dps1) using forward primer 5′-GAAAAAGAGCATATGACCGTC-3′ and reverse primer 5′-CTTCAAGAATTCCCCTTCTCC-3′. The amplified PCR fragment was then cloned into the T7-NT/TOPO vector (Invitrogen). Dps-1 retaining the N-terminal metal site was generated by amplification of the Dps-1 gene lacking 99 bp at the N terminus from pET5a-dps1 using forward primer 5′-GCGGCACCATGCACGCT-3′ and reverse primer 5′-CGTCTTCAAGAATTCCCCTTCTC-3′. The PCR product was then re-amplified using forward primer 5′-CACCATGCACGCTGAC-3′ and reverse primer 5′-CTTCAAGAATTCCCCTTCTCC-3′ to introduce the sequence necessary to clone it into the Champion pET100/D-TOPO vector (Invitrogen). The integrity of the constructs was confirmed by sequencing. Each of the resulting plasmids was transformed into E. coli BL21(DE3)pLysS, and overexpression was induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside at an A600 of 0.3. Cells were pelleted 2 h after induction and stored at -80 °C. The cell pellet was resuspended in a lysis buffer, pH 8.0 (50 mm NaxHyPO4, 300 mm NaCl, 10 mm imidazole, 10% glycerol, 1 mm 2-mercaptoethanol, and 1 mm phenylmethylsulfonyl fluoride), and lysed by sonication. Nucleic acids were digested by the addition of DNase I followed by a 1-h incubation on ice. The cell lysate was centrifuged at 4 °C for 20 min at 5000 rpm. The supernatant was mixed with 5 ml of HIS-Select nickel affinity gel (Sigma) and incubated at 4 °C for 30 min. The mixture was then poured into a column and washed with 5 column volumes of wash buffer, pH 8.0 (50 mm NaxHyPO4, 300 mm NaCl, 20 mm imidazole, 10% glycerol, 1 mm 2-mercaptoethanol, and 1 mm phenylmethylsulfonyl fluoride). Proteins were eluted with a 40-ml linear gradient from 20 mm imidazole (wash buffer) to 250 mm imidazole (elution buffer, pH 8.0, 50 mm NaxHyPO4, 300 mm NaCl, 250 mm imidazole, 10% glycerol, 1 mm 2-mercaptoethanol, and 1 mm phenylmethylsulfonyl fluoride) followed by 50 ml of elution buffer. Pure Dps-dn and Dps-met fractions were pooled, and protein concentrations were determined by quantification of Coomassie Blue-stained SDS-PAGE gels using bovine serum albumin as a standard. Untagged full-length Dps-1 was prepared as described (24Grove A. Wilkinson S.P. J. Mol. Biol. 2005; 347: 495-508Crossref PubMed Scopus (54) Google Scholar). All protein preparations were judged to be >95% pure based on Coomassie-stained SDS-PAGE gels. Cleavage of the His Tag from Recombinant Dps-dn and Dps-met—Fifty μg of protein was incubated with 1 unit of recombinant enterokinase (rEK) from Novagen at room temperature for 16 h in rEK cleavage buffer (50 mm NaCl, 20 mm Tris-HCl, 2 mm CaCl2, pH 7.4) supplied with the enzyme. The cleavage reactions were judged by SDS-PAGE to be complete. Protein Cross-linking—Proteins were cross-linked with 0.1% (v/v) glutaraldehyde in presence of 10 mm Hepes, pH 7.8, and 50 or 500 mm NaCl in a total reaction volume of 10 μl at room temperature for 30 min. The reaction was terminated by the addition of an equal volume of Laemmli sample buffer, and the cross-linked products were analyzed by SDS-PAGE followed by Coomassie Blue staining. Native Polyacrylamide Gel Electrophoresis—The oligomeric state of wild type Dps-1 and the N-terminal deletion mutants was observed on 5% non-denaturing acrylamide gels. The gel recipe was the same as the running gel of SDS-PAGE according to the method of Laemmli, excluding the presence of SDS. The electrophoresis was carried out in 375 mm Tris-HCl, pH 8.7. FPLC and Gel Filtration—All steps of gel filtration were carried out at 4 °C. HiLoad 16/60 Superdex 30 prep grade column (bed length 60 cm, inner diameter 16 mm; GE Healthcare) was first washed with 1 column volume of buffer A, pH 8.0 (50 mm NaxHyPO4, 10 mm imidazole, 10% glycerol) and then with 2 column volumes of buffer B, pH 8.0 (50 mm NaxHyPO4, 300 mm NaCl, 10 mm imidazole, 10% glycerol). The gel filtration standard (Bio-Rad), which is a mixture of bovine thyroglobin (670 kDa), bovine γ-globulin (158kDa), chicken ovalbumin (44kDa), horse myoglobin (17kDa), and vitamin B-12 (1.35kDa), was run to calibrate the column. The concentration of protein applied to the gel filtration column was 5 mg/ml for both wild type Dps-1 and Dps-dn. The proteins were run independently under the same conditions and were eluted with a flow rate of 0.5 ml/min. Ferroxidation by Dps-dn and Dps-met—The kinetics of iron oxidation by Dps-dn and Dps-met was measured at 310 nm using an Agilent 8453 spectrophotometer. Proteins were diluted to 0.2 mg/ml in 20 mm Mops, pH 7.0, 100 mm NaCl. Solutions of ferrous ammonium sulfate, which were used as the source of ferrous iron, were freshly prepared immediately before each experiment. The kinetic data were plotted using Prizm. Electrophoretic Mobility Shift Assays—Supercoiled pGEM5 (100 ng corresponding to 52 fmol of plasmid) was mixed with protein in 10 μl of binding buffer (20 mm Tris-HCl, pH 8, 50 or 500 mm NaCl, 10 mm MgCl2, 0.1 mm Na2EDTA, 1 mm dithiothreitol, 0.05% Brij58, 100 μg/ml of BSA) and incubated at room temperature for 30 min. The entire reaction was then loaded onto a 1% (w/v) agarose gel in 0.5× TBE (45 mm Tris borate, pH 8.3, 1 mm EDTA). The gel was stained with ethidium bromide after electrophoresis. Oligodeoxyribonucleotides used to generate short duplex DNA constructs were purchased and purified by denaturing polyacrylamide gel electrophoresis. The sequence of 26-, 22-, 18-, and 13-bp (all average G + C content) DNA is available as supplemental material. The top strand was 32P-labeled at the 5′-end with phage T4 polynucleotide kinase. Equimolar amounts of complementary oligonucleotides were mixed, heated to 90 °C, and cooled slowly to room temperature (23 °C) to form duplex DNA. Electrophoretic mobility shift assays were performed using 10% polyacrylamide gels (39:1 (w/w) acrylamide:bisacrylamide) in 0.5× TBE unless specified otherwise. Gels were pre-run for 30 min at 175 volts at room temperature before loading the samples with the power on, except for experiments with 18- and 13-bp duplex, which were performed at 4 °C to ensure stability of the duplexes. DNA and protein were mixed in binding buffer (containing 50 mm NaCl for dimeric Dps-1 and 500 mm NaCl for dodecameric Dps-1), and each sample contained 50 fmol (for dimeric Dps-1) or 2.5 fmol (for dodecameric Dps-1) of DNA in a total reaction volume of 10 μl unless indicated otherwise. After electrophoresis, gels were dried, and protein-DNA complexes and free DNA were quantified by phosphorimaging using software supplied by the manufacturer (ImageQuant 1.1). The region on the gel between complex and free DNA was considered as a complex to account for complex dissociation during electrophoresis. Data were fit to the Hill equation, f = fmax[Dps-1]n/(Kd + [Dps-1])n), where [Dps-1] is the protein concentration, f is fractional saturation, Kd reflects the apparent equilibrium dissociation constant, and n is the Hill coefficient. Fits were performed using the program Kaleida-Graph, and the quality of the fits was evaluated by visual inspection, χ2 values, and correlation coefficients. All experiments were carried out at least in triplicate. Effect of Divalent Metal Ions on DNA Binding—To remove the divalent cations from Dps-1, the protein was incubated with 50 mm bipyridyl for 20 min at 4 °C. The bipyridyl or metal-bipyridyl complex was then removed from the protein solution by dialysis against a high salt buffer (10 mm Tris-HCl, pH 8.0, 500 mm KCl, 5% (v/v) glycerol, 0.5 m β-mercaptoethanol, and 0.2 m phenylmethylsulfonyl fluoride) at 4 °C for 2 h. DNA (2.5 fmol) was then incubated with 0–12.4 nm bipyridyl treated protein with or without 80 nm CoCl2 (chosen as it is known from the crystal structure to bind the N-terminal metal site (18Kim S.G. Bhattacharyya G. Grove A. Lee Y.H. J. Mol. Biol. 2006; 361: 105-114Crossref PubMed Scopus (39) Google Scholar)) at room temperature for 30 min. The reactions were analyzed on a 10% polyacrylamide gel (39:1 (w/w) acrylamide:bisacrylamide) in 0.5× TBE. After electrophoresis, the gel was dried, and protein-DNA complexes and free DNA were visualized by phosphorimaging. Netropsin Assay—Eight nm dodecameric Dps-1 was incubated with 50 fmol of 26-bp double-stranded 32P-labeled DNA containing an 8-bp TATA box (the sequence available in the supplemental material) in 10 μl of binding buffer with 500 mm NaCl at room temperature for 45 min. Then the minor groove binding drug netropsin was added to the reaction to a final concentration of 1 μm for an additional 45-min incubation. The effect of netropsin on TATA box binding-protein (TBP)-DNA complex formation was studied in parallel where the same 26-bp duplex DNA was incubated with 1070 pmol of TBP in a reaction buffer (40 mm Tris-HCl, pH 8.0, 10 mm NaCl, 7 mm MgCl2, 3 mm dithiothreitol, 10 μg/ml BSA) followed by the addition of netropsin. All reactions were analyzed on a 10% polyacrylamide gel (39:1 (w/w) acrylamide:bisacrylamide) containing 2.5 mm MgCl2 in 0.5× TBE with 2.5 mm MgCl2. After electrophoresis, the gel was dried, and protein-DNA complexes and free DNA were visualized by phosphorimaging. Methylation Interference Assay—26-bp 32P-labeled double-stranded DNA was methylated by treatment with 0.5% dimethyl sulfate (DMS) for 10 min at room temperature in a total reaction volume of 10 μl. The reaction was stopped by the addition of 2.5 μl of DMS stop solution (1.5 m sodium acetate and 1 m β-mercaptoethanol), and the DNA was recovered by ethanol precipitation. 8 nm dodecameric Dps-1 was incubated with 50 fmol of 26-bp labeled duplex DNA treated with or without DMS in binding buffer containing 500 mm NaCl at room temperature for 60 min. The reactions were analyzed on a 10% polyacrylamide gel (39:1 (w/w) acrylamide:bisacrylamide) in 0.5× TBE. After electrophoresis, the gel was dried, and protein-DNA complexes and free DNA were visualized by phosphorimaging. DNA Cyclization—Plasmid pET5a was digested with BspHI to yield a 105-bp fragment, which was purified on a 2% agarose gel. Ligase-mediated DNA cyclization experiments were carried out with varying protein concentrations. Reactions were initiated by the addition of 80 units of T4 DNA ligase to a final volume of 10 μl. Reactions containing 10–100 fmol of DNA and the desired concentration of Dps-1 or Thermotoga maritima HU were incubated in 1× binding buffer with 200 mm NaCl and 1× ligase buffer at room temperature for 60 min. Reactions were terminated using 3 μl of 10% SDS followed by phenol-chloroform extraction and ethanol precipitation. Reactions were analyzed on a 8% polyacrylamide gel (39:1 (w/w) acrylamide:bisacrylamide) with 0.5× TBE as running buffer. After electrophoresis, gels were dried, and ligation products were visualized by phosphorimaging. All experiments involving protein-DNA interaction or native gel electrophoresis were performed at least in triplicate and with at least two different protein preparations. Structural Considerations—A comparison of the Dps-1 amino acid sequence with that of other Dps proteins reveals that Dps-1 shares significant sequence homology with other Dps homologs, such as complete conservation of residues involved in assembly of the ferroxidase center, and that a main difference is the N-terminal extension (Fig. 1a). The crystal structure of Dps-1 contains ∼177 amino acid residues of each monomer; the first ∼30 residues are not visible, an indication that these residues are freely mobile and may be involved in DNA binding as in the case of E. coli Dps (11Grant R.A. Filman D.J. Finkel S.E. Kolter R. Hogle J.M. Nat. Struct. Biol. 1998; 5: 294-303Crossref PubMed Scopus (445) Google Scholar). The remaining 25 N-terminal residues (Gly-31—Glu-55) preceding the first helix of the four-helix bundle define a loop that harbors a unique metal ion-binding site (Fig. 1b). To specify the role of the N terminus in DNA binding and state of oligomerization of Dps-1, two deletion mutants, Dps-dn and Dps-met, were constructed. Dps-dn lacks the entire 55-amino acid N terminus (thus, comprising residues 56–207), whereas Dps-met lacks only the 33-residue flexible N-terminal region (and comprises residues 34–207). Dps-dn and Dps-met were expressed with an N-terminal His6 tag, whereas wild type Dps-1 is untagged (Fig. 2).FIGURE 2Purified proteins. Coomassie Blue-stained 15% SDS-PAGE gel showing purified proteins. Lane 1, molecular weight markers in kDa; lanes 2–4, 1.5 μg of Dps-dn, Dps-met, and Dps-1, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The N-terminal Metal Site Is Required for Oligomerization— Wild type Dps-1 can exist as a dimer at low salt concentrations, whereas assembly of a very stable dodecamer is favored upon exposure to divalent metals (24Grove A. Wilkinson S.P. J. Mol. Biol. 2005; 347: 495-508Crossref PubMed Scopus (54) Google Scholar). Indeed, the vast majority of recombinant Dps-1 isolated from E. coli is in the dodecameric state as shown by glutaraldehyde-mediated cross-linking (24Grove A. Wilkinson S.P. J. Mol. Biol. 2005; 347: 495-508Crossref PubMed Scopus (54) Google Scholar). In contrast, glutaraldehyde-mediated cross-linking of His6-tagged Dps-dn and Dps-met in buffer containing 50 or 500 mm NaCl yielded dimers as the only cross-linked species, with a significant proportion of protein appearing as monomer (data not shown). The addition of 2 mm MgCl2 or 2 mm ZnCl2 to the cross-linking reactions resulted in the formation of a few higher order oligomers, but dimer remained the predominant species along with uncross-linked monomers. However, removal of the N-terminal domain also removed the majority of the lysines from Dps-dn and Dps-met, which are necessary for glutaraldehyde-mediated cross-linking. To determine rigorously the state of association of Dps-dn compared with Dps-1, FPLC-gel filtration experiments were carried out. From the gel filtration column, the majority of Dps-1 eluted as a high molecular weight oligomer corresponding to Mr ∼ 310, consistent with the dodecamer mass (Fig. 3, b–c). An additional peak appeared at an elution volume of ∼75 ml, indicating the presence of some lower oligomeric species. In contrast, His6-tagged Dps-dn eluted as a single peak of Mr ∼ 44, corresponding to the dimer mass. Consistent results were obtained by native PAGE analysis of Dps-1 and the mutant proteins. On a 5% non-denaturing polyacrylamide gel, His6-tagged Dps-dn, shown by FPLC to exist exclusively as a dimer, migrated as a single band (Fig. 3d, lane 3) with a molecular mass close to BSA (lane 1), whereas dodecameric Dps-1 remained very close to the well of the gel (lane 2). Dps-met, on the other hand, when electrophoresed on a 5% native gel, revealed the presence of two distinct species, one faint lower oligomeric species that migrates near BSA and a larger oligomeric species that runs much higher in the gel (lane 4). The mobility of the lower oligomer of Dps-met is consistent with a dimer mass. Removal of the His6 tag from Dps-dn did not cause assembly of a dodecamer as seen by the even faster migration of the cleaved protein in the native gel (lane 5), whereas His-cleaved Dps-met migrated exclusively as a dodecamer after incubation at room temperature for half an hour in the presence of divalent cation (lane 6), suggesting that the His6 tag of Dps-met hindered proper assembly of the dodecamer. Because the metal is coordinated in part by two histidine residues, these data also suggest that the reason for interference from the vicinal His6 tag is that it impedes proper metal coordination. This interpretation is also consistent with the apparent slower mobility of His6-tagged Dps-met in SDS-PAGE (Fig. 2), which suggests that Dps-1 is more compact than Dps-met due to metal coordination at the N-terminal site. Evidently, retention of the N-terminal metal-binding site is essential for assembly of a dodecameric species. Iron Oxidation Is Not Compromised in Dps-dn and Dps-met— In accordance with the presence of the ferroxidase center, both of the mutant proteins retained the ability to oxidize iron. A progress curve of iron oxidation was measured at 310 nm using His6-tagged proteins. As shown in Fig. 4, upon the addition of five Fe(II) per ferroxidase site, the absorbance gradually increased with time, which implies that Fe(II) was converted to Fe(III) by utilizing the molecular oxygen present in the air. Though Dps-dn was able to oxidize iron, as previously reported for dimeric wild type Dps-1, it did not exhibit significant absorbance at 300–400 nm after the ferroxidation reaction, indicating no mineralized iron core formation (26Macara I.G. Hoy T.G. Harrison P.M. Biochem. J. 1972; 12
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