Name: characterizing dna repair processes at transient and long-lasting double-strand dna breaks by immunofluorescence microscopy
Uploaded: 2018-06-08T13:30:00
Description: Watch this Scientific Journal Video about Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy at JoVE.com

We thank Joel Sanneman and Dr. Philine Wangemann of the Confocal Microscopy Core, funded by the Kansas State University College of Veterinary Medicine, for their support of efforts to develop this technique. pCBASceI was a gift from Maria Jasin (Addgene plasmid # 26477)30. U2OS DR-GFP cells were a kind gift from Maria Jasin18.

Please note, this protocol is written for U2OS cells containing an I-SceI recognition site18. The cells need not be U2OS but must contain the I-SceI site. The protocol may need to be adjusted (e.g., number of cells seeded and incubation times) depending on the type of cells used.
1. Defining the Kinetics of DSB Repair Complex Formation
<ol>
	<li>Grow U20S-DRGFP cells on a 10 cm tissue culture plate until 85&#8211;90% confluent.</li>
	<li>Remove the media and ...

<ol>
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The analysis of DNA damage repair in general and the repair of double-stranded DNA breaks specifically is an active area of research because its consequences span tumorigenesis to basic biology6,20. This manuscript details an approach that accurately dissects the contribution of RAD51 and &#947;-H2AX proteins to the resolution of DSBs through HR. Looking forward, this method can be used to elucidate additional functions of repair proteins at DSBs<sup class="xref"...

Each day, every cell in the human body is bombarded with an estimated 10,000 DNA lesions1. This existential threat puts us at risk for mutations, oncogenesis as well as cell death. To protect genome fidelity, mammalian cells have evolved to respond to DNA damage with a complex series of protein associations and modifications. This response is organized into multiple pathways, collectively known as the DNA damage response (DDR)2,3. The DDR consists of the accumulation of DNA repair proteins at DNA lesions, coordinated both temporally and spatially. DDR frequently induces cell cycle arrest to avoid the propagation or intensification of damage that can occur during the replication of damaged DNA2,4,5. In turn, it is also necessary for the cellular viability to turn off cell cycle arrest by disassociating repair complexes after repair has been completed.
Among the various types of DNA damage, DSBs are the most deleterious. Failure to repair DSBs can result in chromosome rearrangements or large-scale deletions such as the loss of entire chromosome arms. The repair of DSBs is divided into two pathways6,7,8. Homologous recombination (HR) requires a sister chromosome to use as a DNA template and thus is limited to late S and G2/M phases of the cell cycle9,10. Non-homologous end joining (NHEJ) does not have these restrictions but can cause small deletions when repairing DSBs11,12.
DSB repair specifically and the DDR in general are active areas of investigation. Despite being organized into conveniently separated pathways, there is a great deal of redundancy. Indeed, many proteins (BRCA1, BRCA2, and the RPA complex for example) are involved in multiple pathways13,14,15,16. The repair of a lesion by one pathway, can lead to a damage intermediate that must be repaired by another pathway14. The intertwining of these pathways, combined with their complex task of recruiting the right proteins to the correct place for the precise amount of time necessary, requires a multi-tiered regulatory process.
A recent report highlights the intricacies of DDR by demonstrating that repair complex formation, resolution, and localization can each separately be impaired17. The overall goal of the following protocol is to definitively dissect the ability of cells to repair DSB. Using immunofluorescence microscopy, accumulation of repair proteins at the sites of damage can be visualized at the representative time points following the induction of DSBs.
This technique has several advantages to commonly used approaches. Frequently, repair is investigated at single time points and incapable of representing the dynamic process of assembly and dissociation of repair complexes. Observing the full range of repair from the initial activation to the full resolution ensures that a delay in repair is not misidentified as complete inhibition. Conversely, it assures that the induction of a repair response that is unable to inactivate said response is not misidentified as the normal or the excessive activation.
Delayed protein complex formation and the mis-localization of repair proteins, however, may not be unambiguously distinguished with this approach. To determine if repair proteins are mis-localized versus delayed in their localization, a &#34;long-lasting&#34; DSB can be introduced through enzymatic cleavage of cellular DNA. The resulting lesion is recut each time it is repaired, resulting in a distinct large nuclear repair focus and removing the temporal restriction from recruitment. This can be achieved by modifying the existing approach with the use of a rare cutting endonuclease (e.g., I-SceI) to induce a long-lasting DSB. The longevity of DSBs enables the visualization of elusive repair proteins by immunofluorescence microscopy. The enhanced abundance could also improve the visualization when detection is hindered by limitation in the antibody quality, a feature that could be useful when lesser studied proteins are identified as having an impact on DNA repair.
Notably, we provide explicit instructions for a free image processing and analysis software (e.g., ImageJ). This removes a major financial barrier in image analysis, opening the interrogation of DNA damage repair to a wider audience.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12mm Coverslips</td>
			<td>VWR</td>
			<td>89015-725</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (PFA)</td>
			<td>ThermoFisher Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>VWR</td>
			<td>82050-892</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well glass bottom plate</td>
			<td>Cellvis</td>
			<td>P96-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-H2AX Alexa488</td>
			<td>EMD Millipore</td>
			<td>05-636-AF488</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rad51 (D4B10)&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>8875S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-formats plugin for ImageJ</td>
			<td>National Institute of Health (NIH)&#160;</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/plugins/index.html</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>VWR</td>
			<td>97061-416</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>12100046</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15576-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VWR</td>
			<td>89510-194</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG Alexa594</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;A-11012&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>National Institute of Health (NIH)&#160;</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>I-SceI Expression Vector</td>
			<td>Addgene</td>
			<td>26477</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail Polish- Insta Dri</td>
			<td>Sally and Hansen</td>
			<td>Clearly Quick (103)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Bio Basic</td>
			<td>PD8117</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Reagent</td>
			<td>Life Technologies</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TurboFect Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>R0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing dna repair processes at transient and long-lasting double-strand dna breaks by immunofluorescence microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Figure 1 depicts the selection of the correct noise discrimination for maxima/foci quantification using ImageJ. The merged images of DAPI and the repair protein of interest are on the left panel. Figure 1A shows a noise discrimination of 90 and marks the correct number of foci. Nuclei on the edge (depicted with a pink arrow) and foci outside the nuclei (depicted with a yellow arrow) are not counted during the quantification. <str...

Watch this Scientific Journal Video about Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy at JoVE.com

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

This method can help answer key questions in the DNA repair field such as how DNA repair complex formation, localization, and resolution are regulated. The main advantage of this technique is that it can unambiguously detect changes and repair complex localization and kinetics. In addition to these undergraduate researchers in my lab, three graduate students will perform the experiments.That will be Changkun Hu, Dalton Dacus, and Stephen Walterhouse. To begin, grow U2OS/DR-GFP cells on a 10 centimeter tissue culture plate until they are 85 to 90%confluent. Then remove the medium, add three milliliters of EDTA, and incubate the cells at room temperature for three minutes.Replace the EDTA with one milliliter of trypsin and incubate the cells at 37 degrees Celsius for five minutes. Then add five milliliters of DMEM to neutralize the trypsin. After counting the cells, seed six times 10 to the third cells per well in 200 microliters of medium in a glass bottom 96-well plate.Alternatively, seed four times 10 to the six wells in eight milliliters of medium on 12 millimeter cover slips placed in a 10 centimeter plate, then grow the cells over night at 37 degrees Celsius and 5%carbon dioxide. To induce double strand breaks, or DSBs, replace the medium with hydrogen peroxide diluted to a concentration of 25 micromolar with DMEM plus 10%FBS and incubate the plate at 37 degrees Celsius for one hour. Wash the cells three times with 1x PBS, then add fresh DMEM.To fix the cells after incubating them, use PBS to wash the plate three times. Then use a needle and forceps to carefully remove a cover slip from the 10 centimeter plate for each time point and place it in a single well of a 24-well plate. Add 4%PFA and incubate the cells for 15 minutes.Then us PBS to wash the cover slips three times. To permeabilize the cells, add 0.5%non-ionic detergent and PBS. Incubate the cells at room temperature for 15 minutes.Then wash the cells again with PBS three times. Next add 3%BSA and 0.1%non-ionic detergent diluted in PBS to the cover slips in a 24-well plate and incubate the plate at room temperature for one hour. Then add primary antibodies that target the repair protein of interest and incubate the samples at room temperature for one hour.After washing the cells with PBS three times, add DAPI and incubate the plates at room temperature for five minutes. Then incubate the samples with secondary antibodies. After washing the cells as before, use mounting reagent to mount the cover slips cell side down onto slides.Carefully press on the cover slips and use a wipe to soak up any excess mounting reagent. Seal the cover slips with fast drying transparent nail polish and ensure a complete seal of the cover slip with the slide. Take confocal microscope images by focusing on the DAPI channel.To define the nuclei in immunofluorescence images, open the confocal images by dragging the image file into the ImageJ window or by selecting bio-formats importer from the plugin toolbar in ImageJ. Then select split channels. Under color options select colorized from the dropdown menu.Select OK to open the DAPI and histone H2AX images as separate windows and choose the DAPI image. Then select the image toolbar in the adjust threshold option to select the nuclei. Next, adjust the image threshold by sliding the bottom bar of the threshold window completely to the right.Adjust the top bar until the nuclei appear completely red with distinct outlines and the background is black. Then select the analyze toolbar and the analyze particles option. Input the minimum size of the nucleus.From the show dropdown menu select outlines then select the add to manager option. Click OK to open an ROI manager window. From the ROI manager window assign numbers to the nuclei by selection.To use ImageJ to quantify H2AX foci and immunofluorescence microscopy images select the H2AX foci window. Then select process toolbar and choose find maxima to find foci within the nuclei. From the output type dropdown menu choose the single points option and select the preview point selection to visualize the maxima foci.Then input a noise tolerance value depending on the florescence intensity of the image. From the ROI manager window select the nuclei for which H2AX foci must be quantified. Select measure to measure the number of maxima foci within the selected nuclei.Copy the raw intensity values and paste them into software for data analysis. After seating cells on 96-well plates with cover slips as demonstrated earlier in this video, induce double strand breaks by using a lipid-based transfectionary agent to transfect cells with the I-SceI expression vector. To acquire imagine after washing vix permeabilized and blocked cells as shown earlier, add antibodies against gamma H2AX and repair protein of interest.With 1x PBS wash the 96-well plate cover slips three times. Then incubate the 96-well plate cover slips with secondary antibodies. Finally, after identifying the cells that have a large nuclear gamma H2AX focus, manually count those that also show colocalization with the repair protein of interest.This figure shows a noise discrimination of 90 and marks the correct number of foci. Nuclei on the edge and foci outside the nuclei are not counted during the quantification. The panel shown here depicts a low noise tolerance of 50.Numerous maxima are identified outside the nucleus and within the nucleus. A high noise tolerance of 220 is found in this cell. Only a single foci was detected.Colocalization of a gamma H2AX focus and a repair protein of interest is depicted in this figure. An untransfected cell is shown here, and a cell with non-nuclear gamma H2AX focus is shown in the upper right. At higher magnification a clearer picture of a true interior focus and a non-nuclear or exterior focus can be distinguished.Finally, an example of a nuclear gamma H2AX focus without colocalization of a repair protein is shown here. Once mastered, this technique can be done in a few days if it is performed properly. Following this procedure other methods like co-mutor precipitation can be performed in order to identify interactions among repair proteins of interest.After watching this video, you should have a good understanding of how to observer the formation and resolution of DNA repair complexes by inducing transient double strand breaks in DNA. You will also learn how to unambiguously distinguish mislocalization of repair factors from delayed recruitment by inducing long-last lesions.

Research

JoVE Journal

Cancer Research

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