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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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+dynamics+of+the+nonhomologous+end-joining+machinery+during+DNA+double-strand+break+repair.">Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair.</a> Proc Natl Acad Sci. 112 (20), E2575-E2584 (2015).</li><li>Chen, J. J., Silver, D., Cantor, S., Livingston, D. M., Scully, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1,+BRCA2,+and+Rad51+Operate+in+a+Common+DNA+Damage+Response+Pathway.">BRCA1, BRCA2, and Rad51 Operate in a Common DNA Damage Response Pathway.</a> Cancer Res. 59 (7 Supplement), 1752s-1756s (1999).</li><li>D'Andrea, A. 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A., Robinson, K., Howie, H. L., Galloway, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-HPV+5+and+8+E6+Disrupt+Homology+Dependent+Double+Strand+Break+Repair+by+Attenuating+BRCA1+and+BRCA2+Expression+and+Foci+Formation.">&#946;-HPV 5 and 8 E6 Disrupt Homology Dependent Double Strand Break Repair by Attenuating BRCA1 and BRCA2 Expression and Foci Formation.</a> PLOS Pathog. 11 (3), e1004687 (2015).</li><li>Wallace, N. A., Robinson, K., Howie, H. L., Galloway, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPV+5+and+8+E6+Abrogate+ATR+Activity+Resulting+in+Increased+Persistence+of+UVB+Induced+DNA+Damage.">HPV 5 and 8 E6 Abrogate ATR Activity Resulting in Increased Persistence of UVB Induced DNA Damage.</a> PLoS Pathog. 8 (7), (2012).</li><li>Mah, L. J., Vasireddy, R. S., Tang, M. 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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 ...

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Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...