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Analysis of the Repair of
Topoisomerase II DNA Damage

By: Eric Goldstein | Mentor: Dr. Mark Muller

Methods and Materials

NHEJ and HR Reporter Plasmids

The reporter plasmids for both NHEJ and HR employ a similar approach. The presence of the restriction endonuclease ISce-I produces a highly specific DSB at the designated ISce-I cut site (Figure 4). NHEJ or HR can then be used to repair the DSB. With HR, the second cassette has a homologous sequence that allows repair of the DSB, expression of GFP. The NHEJ reporter plasmid has the ISce-I cut site centrally located between the GFP sequence, with GFP expression upon blunt ligation.

Exposure of HR HeLa cells to Etoposide and ISce-I

HR HeLa cells were transfected with pISce-I using Lipofectamine Reagent 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. At 24 hours post transfection, the cells were treated with the desired concentration of VP16 for one hour. Cells were then incubated for 24 and 72 hours in the absence of VP16 to establish a time course for recovery. At the end of each respective recovery incubation time, GFP fluorescence was read using FACSCalibur Fluorescence Activated Cell Sorter (FACS) and CellQuest software (BD Bioscience, San Jose, CA).

Doxycycline inducible ISce-I expression system

The inducible reporter system used for the NHEJ and HR HeLa cells lines (Figure 4) were stably integrated by Dr. Bongyong Lee. The RY-HR HeLa cell line is not doxycycline inducible. These reporters use a tetracycline induction system to transcribe ISce-I endonuclease. Doxycycline is a semi-synthetic tetracycline derivative. Following exposure to doxycycline, the pCMV promoter sequence is exposed, allowing for transcription of the ISce-I downstream gene. Without doxycycline exposure, no ISce-I is expressed. Expression of the ISce-I restriction enzyme creates a DSB at the ISce-I cut site (Figure 4), thereby allowing the cells to perform DSB repair.

Analysis of Repair of Topoisomerase I and II DNA Damage by HR and NHEJ Pathways

Stably integrated RY-HR HeLa cells and inducible, stably integrated NHEJ HeLa cells were observed to be in exponential growth. The plates were then exposed to an increase in VP16 concentration, and an increase in campothecin (CPT), a topoisomerase I poison, for one. A positive control and a transfection efficiency control were set up by the transfection of pISce-I and pEGFP utilizing Lipofectamine 2000 reagent, according to manufacturer's instructions. Single plates for both lines were left untreated and untransfected as a negative control. The plates were then incubated for three days following the drug treatments. After incubation, the supernatant of the plates and the trypsinized cells were transferred to a 15 mL conical tube. The cells were analyzed via FACSCalibur FACS and CellQuest Pro (BD Software) for GFP expression.

Screening of RYHR-GFP clones

HeLa cells stably integrated with the RYHR plasmid (TopoGEN, Daytona Beach, Florida) were provided by Alex Fagenson (Muller lab, UCF). Colonies were selected as single clones and were subcultured to expand the clones. Clone screening was performed by transfecting pISce-I with Lipofectamine Reagent 2000 for five hours. The cells were then incubated in the absence of pISce-I for 24, 72, and 144 hours to establish a time course for recovery. Using the FACSCalibur FACS and CellQuest software, GFP expression was calculated. The best clone was kept for further projects, and the remaining clones were discarded.

Analysis of HR in RY-HR Stably Integrated HeLa Cells Using VP16

Making sure the cells were in heavy exponential growth, increasing VP16 concentrations were added and exposed continuously for five days until 100% confluency. In separate subcultures, a titration of VP16 was exposed to the cells for one hour. One plate was left untreated, and the last plate was exposed to five μg of ISce-I for four hours with Lipofectamine Reagent 2000 according to the manufacturer's instructions in 5 mL OPTI-MEM I. The cells were then grown to confluency without any additives. Using the FACSCalibur and CellQuest software, GFP expression was read.

Trypan Blue Exclusion Assays for Cell Viability

RY-HR HeLa cells and NHEJ HeLa cells were treated with increasing VP16 concentrations. The cells were exposed to VP16 for one hour. After three days incubation without VP16, trypan blue dye was aliquot to microcentrifuge tube. The supernatant of each well was saved in a 15 mL conical tube. The cells were trypsinized and added to the supernatant. From the cell suspension, 20 μL was added to the 180 μL of 0.1% trypan blue dye. From that, 10 μL of the dyed cells were injected into a hemocytometer slide and viewed under a microscope. The slide was divided into four quadrants, and each of the four quadrants was counted and totaled. A ratio of stained versus unstained cells indicates percentage cytotoxicity.

Confocal Microscopy

The three lines of HeLa cells were seeded on a cover slip. ISce-I was transfected using Lipofectamine Reagent 2000 for the non-inducible RYHR clone was added, according to manufacturer's instructions, for four hours, and doxycycline was added to the media for the inducible systems. One hour drug treatments of VP16 were then performed at varying concentrations. Following drug treatment, the cells were incubated for 24 and 48 hours without pISce-I or doxycycline exposure to establish a time course. Using a bent hypodermic needle, the cover slips were carefully removed. A 1.5 μM PI staining solution stained the cells for thirty minutes in the dark. Using FischerFinest Premium Slides, one drop of emulsion oil was placed between the slide and the cover slip. Clear nail polish was then used to seal the cover slip to the slide. Following drying, the slides were analyzed using a confocal microscope at 20X and 100X power.

Figure 4 – The RY-HR and NHEJ Reporter Constructs.

(A): RY-HR reporter system. The RY element and the ISce-I loci create DSBs when exposed to a topoIIα poison or ISce-I restriction endonuclease respectively. Following a DSB, cassette I searches for homology amongst cassette II, leading to HR repair of the DSB and GFP expression. Before HR repair, cassettes 1 and II are incapable of expressing GFP due to mutations in the first cassette and a stop codon in the second cassette. Following HR repair as indicated by the diagram, cassette I can express GFP.
(B): The NHEJ reporter system. The opposing ISce-I restriction cut sites allow for direct ligation of the opposing cassettes initiating GFP expression. The RY sequence is not needed due to the size of the NHEJ reporter system. The RY-HR reporter cassette is substantially smaller than the NHEJ reporter cassette, thus a targeting agent is needed to initiate HR using a topoIIα poison.

Results >>