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20.109(F22): Laboratory Fundamentals of Biological Engineering

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       M1: Genomic instability        M2: Drug discovery        M3: Project design       


The goal of this module is to test the hypothesis that exposure to As inhibits the repair of H2O2-induced DNA damage in an effort to address potential public health risks associated with combined exposures to hazardous compounds. Before we discuss the specific experiments, let's review the important background information.

Hydrogen peroxide (H2O2) is an oxidizing agent
Fa21 guanine and 8-oxo-guanine.png

Normal cell tissues have a basal level of DNA damage due to cell processes involved in cellular metabolism. For example, electrons can escape the electron transport chain and result in the formation of superoxide. Furthermore, defense mechanisms employed to protect the host from bacterial infection involved the release of reactive oxygen species. These reactive oxygen species are implicated in causing more than 20 types of DNA base lesions. One of the most common types of damage is the change of guanine to 8-hydroxyguanine.

Base excision repair (BER) pathway repairs damaged bases
The Base Excision Repair (BER) pathway corrects DNA damage, specifically the removal of small, non-helix distorting lesions in DNA caused by damaged bases. These lesions often result from oxidation, alkylation, deamination, and depuriniation/depryrimidination. If base lesions are not repaired, non-canonical base pairing can occur, which may result in the incorporation of an incorrect base during replication. To prevent mutations and maintain integrity of the genome, the BER pathway evolved as a highly conserved repair mechanism in both E. coli and mammals. Thus, this pathway is responsible for repairing damage before a mutation results.

The core BER pathway includes only four proteins that function to remove the damaged base and replace it with the correct base. First, a DNA glycosylase recognizes that a damaged base is present in the DNA and cleaves an N-glycosidic bond, which creates an apurinic or apyrimidinic site (referred to as an AP site in both cases). Different DNA glycosylases recognize different types of base lesions. Second, the DNA backbone is cleaved to create a single-strand DNA nick by either a DNA AP endonuclease or a DNA AP lyase. Next, a DNA polymerase incorporates the correct base using the sister strand as a template. Last, a DNA ligase completes the repair by sealing the single-strand nick, which restores integrity to the helix. For a more detailed description of the BER pathway, read this review by Robertson et al.

Arsenite (As) inhibits ligase activity
As mentioned above, the final step in BER is a ligation reaction that seals the helix. Arsenite has been shown to enhance the genotoxicity of several mutagens and inhibit ligase activity. Thus, the experiments in this module are designed to test the combined effects of exposure to H2O2, a DNA damaging agent, and As, a chemical known to decrease DNA repair capacity by inhibiting strand ligation.


We will test the hypothesis for this module using two experiments: the γH2AX assay and the CometChip assay. Today we will start the γH2AX experiment. In eukaryotes, including humans, DNA is tightly wound around histone groups. H2AX is a member of the core group of histones that contributes to nucleosome formation and DNA structure. When a DNA double-strand break is introduced into the genome, the H2AX histones near the break are phosphorylated by the ATM kinase at residue Ser-139. Upon phosphorylation H2AX is referred to as gamma-H2AX. Given that only H2AX histones near the site of DNA damage are phosphorylated, γH2AX is a useful target when determining the abundance and location of double-strand breaks. It is important to highlight that the DNA damage expected to occur in response to H2O2 treatment is single-stranded breaks. So why are we using the γH2AX assay to measure double-stranded breaks? When DNA is damaged by multiple single-stranded breaks, double-stranded breaks can occur.

H2AX is phosphorylated in response to DNA double-strand breaks.

Part 1: Define treatment conditions that will be used for γH2AX experiment

The plate you seeded during the previous laboratory session will be used to treat cells using the As and H2O2 conditions detailed below.

Before you complete the protocols for the As and H2O2 treatments, it is important to consider what conditions will be assessed in this experiment and how the experiment was designed. For the experimental question, the goal was to measure DNA damage in cells that were first exposed to As and then treated with H2O2. Given this information regarding the design of the experiment, make a list of all of the conditions that were tested with your laboratory partner. Use the following descriptions of the variables that are included in this experiment to assist you.

  • Three concentrations of As will be tested: 0 μM, 10 μM, and 40 μM
  • Two concentrations of H2O2 will be tested: 0 μM and 1 μM
  • Each concentration of H2O2 will be tested with each concentration of As.

In your laboratory notebook, complete the following:

  • Prepare a list or table of the conditions that will be used for the γH2AX assay.
  • What conditions from your list or table are controls? For what does each condition control?
  • Diagram a basic experimental workflow that illustrates the order in which the treatments will be applied and when the data will be collected.

Part 2: Treat cells for γH2AX assay

For this experiment, the MEF cells will be treated with H2O2 +/- As exposure. The treatments will be applied in two parts. First, cells will be incubated in As for 2 hrs to mimic exposure to the toxic metal. Second, the cells will be incubated in H2O2 for 30 min to induce DNA damage. Following treatment with H2O2 the cells will be visualized to assess DNA damage.

Expose cells to As

  1. Obtain an aliquot of complete media.
  2. Transfer 5 mL of the complete media into three 15 mL conical tubes.
    • One tube will contain media with 0 μM As, one with 10 μM As, and one with 40 μM As. Label the tubes to reflect this information.
  3. Prepare a 10 μM and a 40 μM solution of As-containing media in the appropriate tubes.
    • Stock concentration of As is 10 mM.
  4. Aspirate spent media from all wells in 12-well plate.
  5. Add 1 mL of either 0 μM, 10 μM, or 40 μM As-containing complete media to the appropriate wells according the plate map below.
  6. Incubate cells in the 37 °C incubator for 2 hrs.

In your laboratory notebook, complete the following:

  • Calculate the dilution of As stock needed to prepare a final concentration of 10 μM and 40 μM in 5 mL of media.
    • Stock concentration of As is 10 mM.

Induce DNA damage using H2O2

  1. Obtain an aliquot of serum-free media.
  2. Transfer 7 mL of the serum-free media into two 15 mL conical tubes.
    • One tube will contain media with 0 μM H2O2 and one with 1 μM H2O2. Label the tubes to reflect this information.
  3. Prepare a 1 μM solution of H2O2-containing media in the appropriate tube.
    • Be sure to prepare with serum-free media as the diluent because the serum in complete culture media will inactivate H2O2.
    • Stock concentration of H2O2 is 1 mM.
  4. Retrieve your 12-well plate from the 37 °C and carefully use a P1000 pipet to remove the liquid from each well.
    • Collect the in the As waste container!
  5. Add 1 mL of either 0 μM or 1 μM H2O2-containing media to the appropriate wells according to the plate map below.
  6. Carefully transport your 12-well plate to the 4 °C cooler and incubate for 30 min.

In your laboratory notebook, complete the following:

  • Calculate the dilution of H2O2 stock needed to prepare a final concentration of 1 μM in 7 mL of media.
    • Stock concentration of H2O2 is 1 mM.
F22 gH2AX plate map.png

Part 3: Fix cells for γH2AX staining

  1. Retrieve your 12-well plate from 4 °C following treatment with H2O2.
  2. Aspirate the liquid from the well and immediately add 400 μL of 4% paraformaldehyde to fix the cells.
  3. Incubate at room temperature for 10 min.
  4. Collect the 4% paraformaldehyde in the correct waste stream using a P1000 pipet.
  5. Wash with 500 μL of 1X PBS.
    • Add 1X PBS then remove using a P1000 pipet. Collect the PBS in the correct waste stream.
    • Complete a total of 2 times. After the final wash leave 1 mL of 1X PBS on the cells in the final wash.
  6. Leave all wells with 1 mL 1X PBS, parafilm the sides and move the 12-well plate into the 4 °C cooler.

Reagents list

  • 4% paraformaldehyde (from VWR)
  • arsenite (As) (from Sigma)
  • hydrogen peroxide (H2O2) (from Sigma)
  • phosphate saline buffer (PBS) (from VWR)

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