We are grateful to Michela de Martino, Wilfred R. Hagen, and Kourosh Honarmand Ebrahimi for useful discussions. This work was supported by start-up funding from the Delft University of Technology.

1. Dps Expression and Purification
Obtaining protein of high purity is an essential first step for the DNA-protection assay. Purification of Dps protein can be performed in 4 to 5 days.
<ol>
	<li>Transform a protease-deficient strain of E. coli (such as BL21(DE3) pLysS) with a pET vector (such as pET17) into which the Dps protein-encoding sequence has been cloned.</li>
	<li>Streak the transformed cells out onto Luria Broth (LB) agar plates containing appropriate antibiotics (such as...

<ol>
	<li>Almiron, M., Link, A. J., Furlong, D., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+DNA-binding+protein+with+regulatory+and+protective+roles+in+starved+Escherichia+coli.">A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli.</a> Genes Dev. 6, 2646-2654 (1992).</li><li>Chiancone, E., Ceci, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifaceted+capacity+of+Dps+proteins+to+combat+bacterial+stress+conditions:+Detoxification+of+iron+and+hydrogen+peroxide+and+DNA+binding.">The multifaceted capacity of Dps proteins to combat bacterial stress conditions: Detoxification of iron and hydrogen peroxide and DNA binding.</a> Biochimica et biophysica acta. 1800 (8), 798-805 (2010).</li><li>Ali Azam, T., Iwata, A., Nishimura, A., Ueda, S., Ishihama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+phase-dependent+variation+in+protein+composition+of+the+Escherichia+coli+nucleoid.">Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid.</a> J. Bacteriol. 181 (20), 6361-6370 (1999).</li><li>Grainger, D. C., Goldberg, M. D., Lee, D. J., Busby, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+repression+by+Fis+and+H-NS+at+the+Escherichia+coli+dps+promoter.">Selective repression by Fis and H-NS at the Escherichia coli dps promoter.</a> Molecular Microbiology. 68 (6), 1366-1377 (2008).</li><li>Martinez, A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+DNA+during+oxidative+stress+by+the+nonspecific+DNA-binding+protein+Dps.">Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.</a> J. Bacteriol. 179 (16), 5188-5194 (1997).</li><li>Nair, S., Finkel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dps+protects+cells+against+multiple+stresses+during+stationary+phase.">Dps protects cells against multiple stresses during stationary phase.</a> J. Bacteriol. 186 (13), 4192-4198 (2004).</li><li>Grant, R. A., Filman, D. J., Finkel, S. E., Kolter, R., Hogle, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+Dps,+a+ferritin+homolog+that+binds+and+protects+DNA.">The crystal structure of Dps, a ferritin homolog that binds and protects DNA.</a> Nat. Struct. Biol. 5 (4), 294-303 (1998).</li><li>Zhao, G., Ceci, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+hydrogen+peroxide+detoxification+properties+of+DNA-binding+protein+from+starved+cells.+A+ferritin-like+DNA-binding+protein+of+Escherichia+coli.">Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli.</a> J. Biol. Chem. 277 (31), 27689-27696 (2002).</li><li>Ceci, P., Cellai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+condensation+and+self-aggregation+of+Escherichia+coli+Dps+are+coupled+phenomena+related+to+the+properties+of+the+N-terminus.">DNA condensation and self-aggregation of Escherichia coli Dps are coupled phenomena related to the properties of the N-terminus.</a> Nucleic Acids Res. 32 (19), 5935-5944 (2004).</li><li>Ilari, A., Ceci, P., Ferrari, D., Rossi, G. L., Chiancone, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+incorporation+into+Escherichia+coli+Dps+gives+rise+to+a+ferritin-like+microcrystalline+core.">Iron incorporation into Escherichia coli Dps gives rise to a ferritin-like microcrystalline core.</a> J. Biol. Chem. 277 (40), 37619-37623 (2002).</li><li>Swift, J., Wehbi, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+functional+ferritin-like+proteins+with+hydrophobic+cavities.">Design of functional ferritin-like proteins with hydrophobic cavities.</a> Journal of the American Chemical Society. 128 (20), 6611-6619 (2006).</li><li>Ceci, P., Chiancone, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+iron+oxide+nanoparticles+in+Listeria+innocua+Dps+(DNA-binding+protein+from+starved+cells):+a+study+with+the+wild-type+protein+and+a+catalytic+centre+mutant.">Synthesis of iron oxide nanoparticles in Listeria innocua Dps (DNA-binding protein from starved cells): a study with the wild-type protein and a catalytic centre mutant.</a> Chemistry. 16 (2), 709-717 (2010).</li><li>Ceci, P., Ilari, A., Falvo, E., Chiancone, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Dps+protein+of+Agrobacterium+tumefaciens+does+not+bind+to+DNA+but+protects+it+toward+oxidative+cleavage:+x-ray+crystal+structure,+iron+binding,+and+hydroxyl-radical+scavenging+properties.">The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward oxidative cleavage: x-ray crystal structure, iron binding, and hydroxyl-radical scavenging properties.</a> The Journal of Biological Chemistry. 278 (22), 20319-20326 (2003).</li><li>Su, M., Cavallo, S., Stefanini, S., Chiancone, E., Chasteen, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+so-called+Listeria+innocua+ferritin+is+a+Dps+protein.+Iron+incorporation,+detoxification,+and+DNA+protection+properties.">The so-called Listeria innocua ferritin is a Dps protein. Iron incorporation, detoxification, and DNA protection properties.</a> Biochemistry. 44 (15), 5572-5578 (2005).</li><li>Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+Koelreuteria+paniculata+Laxm.+on+oxidative+stress+and+hydrogen+peroxide-induced+DNA+damage.">Protective effects of Koelreuteria paniculata Laxm. on oxidative stress and hydrogen peroxide-induced DNA damage.</a> Phytopharmacology. 1 (5), 177-189 (2011).</li><li>Stinefelt, B., Leonard, S. S., Blemings, K. P., Shi, X., Klandorf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radical+scavenging,+DNA+protection,+and+inhibition+of+lipid+peroxidation+mediated+by+uric+acid.">Free radical scavenging, DNA protection, and inhibition of lipid peroxidation mediated by uric acid.</a> Annals of Clinical and Laboratory Science. 35 (1), 37-45 (2005).</li><li>Lopez-Gomollon, S., Sevilla, E., Bes, M. T., Peleato, M. L., Fillat, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+role+of+Fur+proteins:+FurB+(All2473)+from+Anabaena+protects+DNA+and+increases+cell+survival+under+oxidative+stress.">New insights into the role of Fur proteins: FurB (All2473) from Anabaena protects DNA and increases cell survival under oxidative stress.</a> The Biochemical Journal. 418 (1), 201-207 (2009).</li><li>Ebrahimi, K. H., Hagedoorn, P. L., Hagen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+and+stimulation+of+formation+of+the+ferroxidase+center+and+the+iron+core+in+Pyrococcus+furiosus+ferritin.">Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin.</a> Journal of Biological Inorganic Chemistry. 15 (8), 1243-1253 (2010).</li><li>Smith, F. E., Herbert, J., Gaudin, J., Hennessy, D. J., Reid, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+iron+determination+using+ferene+triazine.">Serum iron determination using ferene triazine.</a> Clinical Biochemistry. 17 (5), 306-310 (1984).</li></ol>

We have nothing to disclose.

The purification process of Dps as described in this article is very robust. Purity has consistently been high (&gt; 99%); no other proteins appear on SDS-PAGE gels as visible bands. Despite this, some batches of purified Dps appear to have nuclease activity, as evidenced by partial DNA degradation when incubated with very high concentrations of Dps. This might indicate the presence of highly active DNases at low concentration that we were unable to remove through purification. However, this DNA degradation is only obser...

Aerobic organisms must constantly contend with reactive oxygen species that can damage their DNA as well as other crucial biological macromolecules. One potent tool to counteract the toxic effects of oxidative damage is the DNA-binding protein from starved cells (Dps). Since its discovery in 1992 from starved E. coli culture 1, Dps has been identified in more than 300 species of bacteria and archaebacteria 2. Massive upregulation of Dps during stationary phase makes it the most highly expressed nucleoid-associated protein of E. coli under starvation conditions 3, 4. Additionally, Dps has been shown to preserve both bacterial viability and DNA integrity during many various stresses, including starvation, high iron concentration, UV light exposure, heat shock, and oxidative stress 5, 6.
Structurally, Dps self-associates into a stable homo-oligomeric complex of 12 monomers, which assemble into a spherical hollow shell. The ~4.5 nm-wide internal cavity is accessible to the exterior solvent via pores that allow the passage of small molecules 7, and it can sequester mineralized metals such as iron 8. The protective effect of Dps derives from its several biochemical activities, which include non-specific DNA binding 1, ferroxidase activity, and iron storage 8.
Detailed study of the beneficial biochemical activities of Dps first requires its purification. Dps purification is an elaborate procedure, as Dps must be separated not only from other proteins, but also from any bound DNA 7. Our optimized purification process uses many common techniques, consisting of two ion-exchange columns and an ammonium sulfate precipitation step. Several buffer exchanges are needed, as highly concentrated Dps can precipitate out of solution in low salt conditions. Once Dps protein has been purified, it may be applied to assays that directly measure its ferroxidase activity 8, DNA-binding stoichiometry 9, and mechanisms of iron binding 10. Purified Dps also has other potential applications. The stable hollow spherical structure of Dps has been used as scaffolding for storing hydrophobic particles inside the protein cavity 11 and even as a reaction chamber to synthesize novel magnetic nanoparticles 12.
The protective ability of Dps to mediate damage due to reactive oxygen species can be clearly and directly demonstrated using the DNA protection assay 13, 14. In this in vitro procedure, radical species are produced when iron catalyzes H2O2 degradation through Fenton chemistry. These radicals directly damage DNA present in the reaction and can completely degrade it at high concentrations. Two key Dps activities may both directly counteract the effects of Fenton-mediated radical production. Dps lowers the concentration of catalytic iron through mineralization, consuming the available hydrogen peroxide in the process. Additionally, Dps binding to DNA may potentially shield it physically from radical damage and condenses it into a smaller volume with less reactive surface area. The combination of these two properties makes the DNA protection assay well suited for the purpose of measuring protective Dps activity.
The DNA protection assay is quite versatile and can be used for a variety of applications beyond Dps characterization. Radical damage is a common form of stress in cells, and many different proteins and chemicals are used to counteract it. The general principle of the assay, using DNA integrity as a marker for radical damage, can be used in combination with almost any radical-producing reaction or counteracting agent. Among others, the assay has been successfully used to determine anti-oxidative properties of K. paniculata extract for use in food industry 15, to characterize the effects of uric acid on hydroxyl damage mediation 16, and to gain new insights into the function of Fur transcriptional regulator proteins 17.
Despite the numerous uses of the assay in published papers, we found that many optimization and troubleshooting steps were required, which makes setting up the assay for the first time an unnecessarily laborious process for many researchers. The protocol we present in this article aims to remove this barrier for entry.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>BL21(DE3)pLysS competent cells</td>
 <td>Promega</td>
 <td>L1195</td>
 </tr>
 <tr>
 <td>pET-17b DNA</td>
 <td>EMD-Millipore</td>
 <td>69663-3</td>
 </tr>
 <tr>
 <td>LB broth powder</td>
 <td>Sigma-Aldrich</td>
 <td>L3022</td>
 </tr>
 <tr>
 <td>Ampicillin sodium salt</td>
 <td>Sigma-Aldrich</td>
 <td>A0166</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>C0378</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Sigma-Aldrich</td>
 <td>I6758</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>BDH</td>
 <td>441476L</td>
 </tr>
 <tr>
 <td>Potassium hydroxide</td>
 <td>Merck</td>
 <td>105033</td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>VWR</td>
 <td>443824T</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>E9884</td>
 </tr>
 <tr>
 <td>Protease Inhibitor Cocktail Set III</td>
 <td>EMD-Millipore</td>
 <td>539134</td>
 </tr>
 <tr>
 <td>DEAE-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>DFF100</td>
 </tr>
 <tr>
 <td>Ammonium sulphate</td>
 <td>Sigma-Aldrich</td>
 <td>A4418</td>
 </tr>
 <tr>
 <td>PD-10 Desalting Columns</td>
 <td>GE Healthcare LS</td>
 <td>17-0851-01</td>
 </tr>
 <tr>
 <td>SP-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>S1799</td>
 </tr>
 <tr>
 <td>Amicon Ultra Centr. Filter (10K MWCO)</td>
 <td>Millipore</td>
 <td>UFC901024</td>
 </tr>
 <tr>
 <td>Ferrous Sulphate heptahydrate</td>
 <td>Sigma-Aldrich</td>
 <td>F8048</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide solution</td>
 <td>Sigma-Aldrich</td>
 <td>216763</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Calbiochem</td>
 <td>475898</td>
 </tr>
 <tr>
 <td>SDS solution</td>
 <td>Bio-Rad</td>
 <td>161-0418</td>
 </tr>
 <tr>
 <td>Ethidium bromide</td>
 <td>Sigma-Aldrich</td>
 <td>E1510</td>
 </tr>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Static incubator</td>
 <td>Hettich</td>
 <td>INE500</td>
 </tr>
 <tr>
 <td>Shaking Incubator </td>
 <td>New Brunswick Sc.</td>
 <td>Inova 44</td>
 </tr>
 <tr>
 <td>Cooled centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Avanti J-E</td>
 </tr>
 <tr>
 <td>Table-top centrifuge</td>
 <td>Eppendorf</td>
 <td>5424</td>
 </tr>
 <tr>
 <td>Cell disrupter</td>
 <td>Constant Systems Ltd.</td>
 <td>TS2/40/AA/AA</td>
 </tr>
 <tr>
 <td>FPLC Purifier</td>
 <td>General Electric</td>
 <td>AKTA</td>
 </tr>
 <tr>
 <td>Airtight vials</td>
 <td>Cole-Parmer</td>
 <td>EW-08918-85</td>
 </tr>
 <tr>
 <td>Syringe needles</td>
 <td>BD</td>
 <td>305128</td>
 </tr>
 <tr>
 <td>Pipettes</td>
 <td>Eppendorf</td>
 <td>Z683779-1EA, Z683795-1EA</td>
 </tr>
 </tbody>
</table>

application of an in vitro dna protection assay to visualize stress mediation properties of the dps protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

The purification process for Dps described here is very reproducible. Dps purification according to the described protocol, using 2 L of E. coli culture as a starting point, will typically yield 2.5 ml of protein containing Dps12 at concentrations between 5 and 12 &mu;M. Longer induction times (4 hr) seem to reduce this variability. Protein purity is consistently above 99%, as evidenced by SDS-PAGE gels (Figure 1). The level of DNA contamination is consistently negligible, as evidence...

Watch this Scientific Journal Video about Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein at JoVE.com

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

application-an-vitro-dna-protection-assay-to-visualize-stress

Research

JoVE Journal

Biology

11.1K visualizações.  Delft University of Technology. A proteína de ligação ao DNA de células famintas (SAD) desempenha um papel crucial no combate ao stress bacteriana. Este artigo discute a purificação da E. coli Dps eo protocolo para um In vitro Ensaio demonstrando proteção Dps mediada por DNA da degradação pelas espécies reativas de oxigênio.