20.109(F17):Test role of biochemical factors in genomic stability (Day4)

Introduction

In the initial experiment for this module, your goal was to optimize the CometChip assay. Now that you have data concerning how to best load cells into the CometChip, you will use this technique to study the Base Excision Repair (BER) pathway. BER corrects DNA damage, specifically base lesions, that 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.

Base excision repair pathway.

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.

The BER pathway repairs base lesions using either a short-patch or long-patch mechanism. In the short-patch BER pathway, a single nucleotide is removed in the repair process. In contrast, in the long-patch BER pathway at least two nucleotides are removed. Though both mechanisms of repair are utilized, the conditions that drive each are poorly understood.

To study the BER pathway, as a class you will use two chemical treatments to generate base lesions. The treatments will be applied to two CometChips as a second variable will be introduced during the next laboratory session.

mutant cell lines!!!

Protocols

Part 1: Load CometChips to test biochemical factors

1. You will load the CometChips you prepared during the previous laboratory session using the procedure from M1D2.
   • For the cell number, you will use the values decided on during the group discussion on M1D3.
   • The cell suspension prepared by the teaching faculty is at a density of XX cells / mL.

Include information re: different cell types

Part 2: Induce DNA damage using H₂O₂ or MMS treatments

After you load the cells into the CometChip, you will induce DNA damage using either H₂O₂ or MMS. Each team will select one of the chemical treatments on the sign-up sheet located at the front laboratory bench.

Follow the appropriate protocol for damage induction based on the chemical you selected for your experiment.

DNA damage inducer: H₂O₂
You will use the following procedure to treat both of your CometChips.

1. While the LMP agar is solidifying at 4 °C, calculate the dilutions of H₂O₂ that you will use for your experiment.
   • The H₂O₂ stock solution is at a ~10 M concentration.
   • You will add 100 μL per well to induce DNA damage.
   • Calculate dilutions that result in doses of 0, 5, 10, 15, 20, and 25 μM.
2. Retrieve your CometChip from the 4 °C and carefully replace the bottomless 96-well plate to recreate the wells that you used for cell loading.
   • Secure the bottomless 96-well plate with binder clips as before.
3. Obtain an aliquot of H₂O₂ and prepare the doses that you calculated in Step #1 using 1x PBS as the diluent in a 12-well reservoir as discussed in prelab.
   • Be sure to keep the H₂O₂ on ice at all times!
4. Using a multi-channel pipet, add the appropriate treatment doses of H₂O₂ to each well of your CometChip.
   • The 0 μM treatment should be added to the wells at the far left and the 80 μM treatment should be added to the wells at the far right of the CometChip.
5. Carefully transport your CometChip to the 4 °C cooler and incubate for exactly 20 min.
6. Retrieve your CometChip from the 4 °C cooler and quickly remove the bottomless 96-well plate while holding the CometChip at an angle such that the low doses of H₂O₂ 'run' over the wells with high doses.
   • If the high doses of H₂O₂ run over the low dose wells, additional damage may be induced and confound the results.
7. Rinse your CometChip using 1x PBS as done during the cell loading procedure.
   • Complete a total of 3 washes.

DNA damage inducer: MMS
You will use the following procedure to treat both of your CometChips.

Please be very careful when using MMS as it is a mutagen. You should always wear flock-lined gloves, in addition to the standard laboratory gloves, when using MMS.

1. While the LMP agar is solidifying at 4 °C, calculate the dilutions of MMS that you will use for your experiment.
   • The MMS stock solution is at a ~12 M concentration.
   • You will add 100 μL per well to induce DNA damage.
   • Calculate dilutions that result in doses of 0, 0.1, 0.2, 0.5, 6, and 8 mM.
2. Retrieve your CometChip from the 4 °C and carefully replace the bottomless 96-well plate to recreate the wells that you used for cell loading.
   • Secure the bottomless 96-well plate with binder clips as before.
3. Obtain an aliquot of MMS and prepare the doses that you calculated in Step #1 using 1x PBS as the diluent in a 12-well reservoir as discussed in prelab.
   • Work on at the piece of diaper paper located at the front laboratory bench when preparing MMS solutions!
4. Using a multi-channel pipet, add the appropriate treatment doses of MMS to each well of your CometChip.
   • The 0 mM treatment should be added to the wells at the far left and the 8 mM treatment should be added to the wells at the far right of the CometChip.
5. Carefully transport your CometChip to the 37 °C incubator for exactly 30 min.
6. Retrieve your CometChip from the 37 °C incubator and quickly remove the bottomless 96-well plate while holding the CometChip at an angle such that the low doses of MMS 'run' over the wells with high doses.
   • If the high doses of MMS run over the low dose wells, additional damage may be induced and confound the results.
7. Rinse your CometChip using 1x PBS as done during the cell loading procedure.
   • Complete a total of 3 washes.

Part 3: Complete cell lysis

1. Obtain a fresh dish and an aliquot of alkaline lysis buffer from the front laboratory bench.
2. Carefully move your CometChip into the dish.
3. Add the aliquot of alkaline lysis solution by slowly pouring it into a corner of the dish rather than directly over the CometChip.
   • Be sure the CometChip is completely submerged and not floating in the dish.
4. Label the dish with your team and section information.
5. Move your dish into the 4 °C cooler.

The teaching faculty will transfer your CometChip from the alkaline lysis solution into 1X PBS at ~24 hr.

Reagents list

Navigation links

Next day: Complete biochemical experiment

Previous day: Evaluate cell loading results and prepare biochemical experiment