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Biology

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

Published: May 31st, 2013

DOI:

10.3791/50390

1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology

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.

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.

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 ba....

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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.

  1. 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.
  2. Streak the transformed cells out onto Luria Broth (LB) agar plates containing appropriate antibiotics (such as.......

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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 μM. 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.......

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The purification process of Dps as described in this article is very robust. Purity has consistently been high (> 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.......

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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.

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

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