Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The overall goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including simple strand breaks, and complex damage, to study DNA damage signaling and repair factor assembly in vivo. In the 1960s and 70s, we focused a laser beam through a microscope that you can see behind me, and was able to damage a small region of a chromosome. The cell divided, survived, and we couldn't understand why.

Well, 40 years later, we are now using this method to selectively study DNA repair, and that's what you're going to see today. This method can help answer key questions in the DNA repair field, such as how DNA damage is recognized and processed and how damage signal propagates in the cell. Laser microirradiation allows for real time, high resolution, single cell analysis of macromolecular dynamics in response to DNA damage confounded to a several micrometer region within the cell nucleus.

However, various laser conditions have been used without appreciation for differences in the types of damage induced, causing inconsistencies in cellular responses, factor recruitment and modifications. To express fluorescently-tagged proteins in Hela cells, on day one, seed six to eight times 10 to the fourth cells in 0.5 milliliters of culture medium in one well of a 24-well plate. Then place the plate in the tissue culture incubator.

On day two, perform liposome-mediated DNA transfection by diluting 0.3 micrograms of a mammalian expression plasmid DNA encoding a fluorescently tagged DNA repair factor into 25 microliters of serum-free culture medium. Here, we use the damage site targeting domain of 53BP1 fused to GFP for double strand break damage, or DNA glycosylase NTH1 fused to GFP for base damage. In another 1.5 milliliter tube, dilute one microliter of liposome-based DNA transfection reagent into 25 microliters of serum-free culture medium, then mix the content of two tubes, diluted DNA and transfection reagent together and incubate the tube at room temperature for 10 minutes to form DNA-lipid complexes.

Rinse the cells once with 0.5 milliliters of culture medium, then remove the medium and add 0.5 milliliters of fresh culture medium to the cells. Add the DNA-lipid complexes to the cells and gently mix by rocking the plate a few times. Then return the cells to the incubator.

After four to six hours, remove the culture medium from the cells, and add 300 microliters of 0.5%Trypsin-EDTA. Remove the solution, then add 200 microliters of Trypsin-EDTA and incubate the cells at 37 degrees Celsius for three to four minutes. After confirming that the cells are detached, add 800 microliters of culture medium and resuspend the cells by gently pipeting to break up the cell clumps.

Transfer the cell suspension to a 35-millimeter culture dish with a gridded coverslip, and add culture medium to a final volume of two milliliters. After 48 hours, the cells will be at 30 to 60%confluency with fluorescent protein expressed, and ready for laser damage induction. Turn on the NIR laser and allow the laser system to warm up for one hour before use.

Place a dish of cells on a 37 degree Celsius heated stage in a chamber that allows carbon dioxide and humidity control. For live-cell analysis of fluorescently tagged proteins, use a high magnification oil immersion objective and an appropriate fluorescence filter combination depending on the tag. Search for cells that have comparable fluorescent protein expression.

Avoid cells that have too weak or too strong expression in order to reduce cell-to-cell variability in fluorescence measurements. For confocal microscopy, use a 100x 1.3 numerical aperture oil objective lens and the software bleach function to target linear tracks inside the cell nucleus. Adjust the laser transmission percentage to 5%without changing any other settings.

Then place a cell in the center of the field. Click the region of interest, and using the mouse, draw a line or box in the nucleus approximately 50 by four pixels. Avoid touching the nuclear membrane or nucleoli.

Then click the bleach button to induce DNA damage. Here, the difference between the peak recruitment of GFP-53BP1 and GFP-NTH1 are evident. To carry out quantitative fluorescence analysis of live cells with the confocal microscope, use 1x magnification, and an argon laser for GFP FITC.

Set a resolution of 512 x 512 pixels, a scan speed of five, and number one for quick scan or two plus for averaging over multiple scans to improve signal-to-noise ratio. Then capture images. Draw the bleach ROY for damage induction, then select the acquired Time Series sub menu, set number at 20, and the cycle delay at 30 seconds.

Choose bleach once after first scan, and then click start B.In this case, the first image is acquired immediately before damage induction, followed by the acquisition of 19 additional images at 30 second intervals. Here, you can see that the GFP-NTH1 is rapidly and transiently recruited to damage sites, whereas GFP-53BP1 accumulates slowly, and persists at damage sites. Some repair factors may be recruited to damage sites in a cell cycle specific manner.

To identify G1 phase cells for damage induction, under an inverted microscope, using a 10x objective, and 10x ocular lenses, identify metaphase cells which are slightly lifted and rounded in shape. Acquire images to record the position of metaphase cells on the gridded coverslip. After three to four hours, confirm cell division has resulted in two daughter cells that are ready for damage induction at the recorded position.

To prepare SG2 phase cells, use a double thymidine block protocol. Confirm synchronization efficiency by DNA profiling and or detection of Rad51 at damage sites. At high-input power laser damage, clustering of CPD UV damage and GFP-NTH1 DNA glycosylase that specifically recognizes base damage were more clearly observed compared to low input power.

In addition, higher levels of XRCC1 and CtIP were observed, reflecting the increased number of single and double strand breaks, and thus indicating that complex DNA damage was induced. As demonstrated here, variation of laser input power results in changes in damage signaling. PARP1 mediates poly ADP-ribosylation or PAR of target proteins at damage sites which is significantly induced by high input power laser but only weakly by low laser power.

53BP1 is recruited to low, but not high input power damage sites. Interestingly, the recruitment to low input power damage was suppressed by the presence of the second high input power damage in the same cell nucleus. Inhibition was alleviated by the PARP Inhibitor.

Conversely, the inhibitor of PARP that breaks down PAR, enhanced the PAR signal, and effectively suppressed 53BP1 recruitment even at the low input power damage sites. This indicates that PARP signaling, and not the nature of the damage, per say, dictates 53BP1 recruitment. This is an example in which laser titration revealed differential activation of PARP1 gauging the amount and complexity of DNA damage, and controlling the damage site accessibility.

Once mastered, this technique can be done in about five days, including cell preparation, laser system setup, DNA damage induction, imaging of live or immunosilent cells, and the data analysis. It may take a longer with cells synchronization or time cost analysis. While attempting this procedure, it's important to remember that titration of laser input power may deviate over time, even in the same laser system, especially after system realignment, thus it is important to periodically check the repair factor recruitment pattern.

Following this procedure, results from other DNA damaging methods, such as analyzing irradiation, chemical agents, or expression of endonucleases, can be performed and compared in order to confirm that the results are not due to laser specific artifacts. After it's development, this technique paved the way for researcher's in the field of DNA repair to explore the DNA damage response with regard to nuclear chromatin, and to identify new factors and pathways of DNA damage, recognition, processing, and signaling.