The overall goal of this procedure is to perform single molecule analysis of the distribution of laser localized interstrand crosslinks in genomic DNA in order to study cellular responses to DNA interstrand crosslinks. This method can help answer key questions in the DNA repair field, regarding the distribution of DNA lesions introduced by laser technology, a widely used experimental tool. The main advantage of this technique is that it allows direct imaging of DNA lesions introduced by the laser activated compound in genomic DNA.
This has not been possible before. Demonstrating the procedure will be Julia Gichimu, and Hima Gali from our lab. Every time the targeting laser is used it is important to perform a mirror slide calibration to verify that the laser is hitting at the appropriate XYZ coordinates, and to establish the area of the field captured by the camera where the laser can be set to strike.
Find a field on the slide that has been previously etched. Set the exposure time and laser power. Then adjust the focus to move to a nearby non targeted field on the mirror slide.
Start the calibration procedure. The software will fire the laser to etch the mirror slide along horizontal and diagonal line. These lines define the boundaries of the field in which the laser can strike a target cell.
Set several regions of interest, or ROIs, inside the field and fire the laser to confirm that it is targeting the selected areas and thus striking at the appropriate XYZ coordinates. A biological calibration must also be performed when setting up the microscope targeting laser system. The first step is to define the minimum laser power required to photoactivate trimethylpsoralen, or TMP, induce interstrand crosslinks, and provoke recruitment of a GFP tagged repair factor, for which kinetics can previously be characterized.
In this laboratory a U2OS cell line stably transfacted with a plasmid expressing GFP fan one is used. As shown here, when cells are exposed to six micromolar TMP and 1.2%laser an image for 10 minutes, viewing the sequence of images as a video confirms the recruitment of GFP fan one to the targeted ROI. The second step is to verify that this power setting does not trigger recruitment in the absence of the compound.
As shown here, when cells are exposed to 1.2%laser alone image for 10 minutes and the image is viewed as a video there is no recruitment of GFP fan one to the targeted ROI. Thirdly, it is important to define the minimum power setting that is able to blow out a cell. The ability to blow out a cell at this power setting should be tested as a routine biological calibration every day the targeting laser is used.
Set the ROI inside the nucleus while avoiding the nucleoli. Adjust the focus. Set the laser power to 12%and fire.
Finally, it is essential to set the focal plane at mid cell to achieve correct targeting. To demonstrate this three cells are targeted at three focal planes. Mid cell.
Too high. And too low. Three minutes after targeting a stack of images spanning three microns is generated to examine the distribution of the GFP fan one stripe throughout the imaged focal plane.
Only the cell targeted at mid cell shows a GFP fan one stripe in all Z planes. Prior to performing laser localization prepare a 35 millimeter glass bottom cell culture dishes for growing cells. With a marking pen make a vertical mark on the side of each dish.
Use a diamond pen to scratch a cross in the center of the glass on the culture surface, such that the black stripe on the side of the dish is at the top of one arm of the cross. The cross is marked on the growth surface of the glass so that it and the cells will be in the same focal plane. Plate cells and sterilized cross marked culture dishes and incubate for 24 hours in one micromolar five chloro deoxyuridine to uniformly label DNA.
On the day of the experiment, add digoxigenin linked TMP or dig TMP in 50%ethanol to the cell culture medium to a final concentration of 20 micromolar. Bring the medium to 37 degrees Celsius. Replace the medium in the culture dishes with dig TMP containing medium and incubate at 37 degrees Celsius and 5%CO2 for 30 minutes to allow the dig TMP to equilibrate.
While the cells are incubating perform mirror slide calibration and 12%blow out of a cell as demonstrated earlier. Place the plate in the environmental chamber at 37 degrees Celsius with controlled CO2 and humidity on the microscope stage. Focus with the 60X oil objective on the intersection of the cross.
Select the first field to target and use the shape tool to set the ROIs in individual cells located in the immediate vicinity of the intersection of the cross. Select nucleoplasmic areas outside the nucleoli. Adjust the Z focal plane to target the laser midway through the nuclei.
Next set the power setting to the minimum required to activate the psoralen in the interstrand crosslink induced DNA damage response. 1.2%is used here. Fire the laser.
After all cells in a field have been targeted, move the plate to a new field, staying close to the intersection of the cross. Repeat the procedure just demonstrated in 12 to 18 fields to target a total of 80 to 100 cells. To harvest the cells, remove the culture dish from the stage.
Remove the medium and wash the cells with PBS solution. Remove the PBS and place a 10 microliter drop of a commercial trypsin EDTA solution on the intersection of the cross in the middle of the glass surface. Incubate for three to four minutes at room temperature and then draw the trypsin solution with detached cells into a pipette tip.
Place the drop of trypsin EDTA with the cells at one end of silanized glass microscope slide. To lyse the cells and release the DNA add 10 microliters of 5%SDS solution and incubate for three to four minutes at room temperature, mixing gently with pipette tip, allowing the periphery of the pool to dry. Tilt the slide 20 degrees and allow the liquid to run down to the end.
The hydrodynamic forces of the flowing liquid will extend and stretch the DNA fibers. Let the slide air dry for 10 minutes. Immerse the slide in a solution of three to one methanol to acetic acid and fix for 10 minutes.
Then remove the slide from the fix solution and air dry again. At this point the slide can be stored indefinitely in 70%ethanol at 20 degrees Celsius. To process the slide for detection of dig signals on DNA fibers by fluorescence microscopy remove the slide with the stretched DNA from the 70%ethanol solution and allow it to air dry.
Subsequently denature the DNA fibers in hydrogen chloride and then transfer them to a container of tris hydrochloridic at PH eight for neutralization. Next wash the samples in PBS. And finally, stain them with the appropriate antibodies following a previously published procedure.
The structure of trimethylpsoralen with a dioxigenin tag is shown. Interstrand DNA crosslinks, or ICLs, can be displayed by immuno florescence against dig TMP. This example shows cells with an accumulation of a well known marker of the DNA damage response gamma H2AX, visualized in green, in ROIs containing laser localized ICLs visualized in red.
The DAPI stained nucleus is in blue. The bright field shows a cell partially on the mark made by the diamond pen. Note that ROIs are placed outside the nucleoli.
To determine the frequency and spacing of adducts cells were incubated in five chloro deoxyuridine for 24 hours prior to introduction of the ICLs. After harvesting the cells and spreading the DNA fibers the dig signals were detected with an amino quantum dot visualized in red in the five chloro deoxyuridine by amino florescence visualized in green. Analysis of the dig signals on the fibers reveal that the inter ICL distances range from less than 10 KB to greater than 160 KB.There was no evidence for clustered adducts.
Following these guidelines it should be possible to extend this approach to other DNA adducts, such as oxidative lesions that can be localized by lasers of appropriate wavelength.
