We thank Dr. Akira Yasui at Tohoku University, Japan for the GFP-NTH1 expression plasmid, and Dr. Eros Lazzerini Denchi at the Scripps Research Institute, La Jolla, California for the TRF2-YFP and EGFP-53BP11220-1711 expression plasmids. This work was supported by the Air Force Office of Scientific Research (FA9550-04-1-0101) and the Beckman Laser Institute Inc. Foundation (to M.W.B), the Ford Foundation Fellowship from the National Academy of Sciences (to B.A.S), and NSF MCB-1615701 and CRCC CRR-17-426665 (to K. Y.).

1. Basic Cell Preparation
Note: This step is for standard immunofluorescence detection of the endogenous protein recruitment or modification, and for the use of a cell line stably expressing a fluorescently tagged recombinant protein. For example, Potorustridactylus (PtK2) marsupial kidney epithelial cells stably expressing EGFP-53BP11220-1711 or TRF2-YFP were used in our previous study (Figure 1)32. In the former case, th...

The advantages of using laser microirradiation for DDR research are:
1. It is feasible to induce different types and amounts of DNA damage from simple strand breaks to complex DNA damage, and to detect different repair factors at the DNA damage sites by adjusting the laser irradiation parameters. It is also possible to inflict damage multiple times in the same cell nucleus in order to evaluate trans effects (as in Figure 3).
2....

In Vivo DNA Damage Signaling is Not Well Understood 
In vivo, DNA is complexed with histones and other factors to form chromatin fibers. Regulation of chromatin structure is of paramount importance for DNA metabolism. For example, the histone variant H2AX is phosphorylated by ataxia-telangiectasia mutated (ATM) and other kinases following double-strand breaks (DSB) induction, and is important for DSB damage signal amplification as well as providing a docking site for other factors. Spreading of damage signaling and repair pathway choice appear to be critically influenced by local chromatin structure at damage sites1. A number of chromatin remodeling factors, histone chaperones, and histone modifying enzymes are indeed recruited to damage sites and are important for efficient DNA repair, highlighting the significance of chromatin regulation in the DDR and repair2,3,4. Furthermore, damage site clustering or repositioning was observed in yeast and drosophila5,6,7,8, reminiscent of relocalization of gene loci in the subnuclear compartment associated with gene regulation9,10. Recent studies in mouse and human cells also revealed mobilization of DSB sites, which influences repair fidelity and pathway choice11,12. This raises the possibility that DDR/repair may also be intimately linked to nuclear architecture, higher-order chromatin organization, and chromosome dynamics in the cell nucleus. Thus, it is critical to develop high-resolution methods to study DDR and repair processes in the context of the endogenous nuclear environment in a live cell in order to understand short- and long-term consequences of DNA damage.
Critical Role of PAR Polymerase (PARP) in Gauging the Extent and Type of Damage and Regulating Protein Assembly at the Damage Site 
PARP1 is a DNA nick sensor rapidly activated by DNA damage that plays a critical role in DNA repair13. PARP1 was originally thought to function together with X-Ray Repair Cross Complementing 1 (XRCC1) to facilitate base excision repair (BER), but recent studies reveal its role in other DNA repair pathways, including DSB repair14. Activated PARP1 uses nicotinamide adenine dinucleotide (NAD+) as a substrate to ADP-ribosylate multiple target proteins, including itself. This enzyme and other family members have attracted much attention in recent years as PARP inhibitors have emerged as promising therapeutic drugs for cancers. Although PARP inhibitors were initially found to be effective in breast cancer gene (BRCA)-mutated breast cancer cells, there is now a plethora of evidence for their effects in mono- and combination therapies together with DNA damaging agents/irradiation against a broad spectrum of cancers with mutations not limited to BRCA15,16,17,18,19,20.
At the molecular level, PARP activation was shown to play critical roles in organizing the local chromatin structure at damage sites. PAR-dependent recruitment of chromatin modification enzymes facilitates DSB repair and dictates repair pathway choices, suggesting the important scaffolding role of PAR modification at damage sites. 13,21,22,23,24,25,26,27,28,29,30,31 We recently demonstrated the exclusion of p53-binding protein 1 (53BP1) from damage sites by PAR 32, providing an alternative explanation for 53BP1-dependent hyperactivation of non-homologous endjoining (NHEJ) by PARP inhibitor and highlighting the significance of PARP in DSB repair pathway choice33,34. PARP1 also directly PARylates and affects the activity of multiple DNA repair factors14.
Using Laser Microirradiation as a Tool to Study DDR/Repair In Vivo 
Laser microirradiation to produce sub-micron alterations on individual chromosomes was first described in 196935 and reviewed in detail in 198136. Several decades later, laser microirradiation was shown to induce DNA damage at a defined submicrometer region in the cell nucleus, and was proven to be a valuable technique to study recruitment or modifications of various factors to DNA lesions in vivo13,37,38,39,40,41. This method allows detection of those factors that do not form distinct irradiation-induced foci (IRIF) at damage sites39,42. It is also possible to examine the spatiotemporal dynamics of chromatin structural changes both at damage sites and in the rest of the nucleus. We carefully compared the DDRs induced by different laser systems and input powers to evaluate the relationship between the type of DNA damage and the microirradiation conditions32,43,44,45. The aberrant recruitment patterns of 53BP1 and telomeric repeat binding factor 2 (TRF2) were observed in the previous laser damage studies, which provided the basis for the recurring concern for the &#34;non-physiological&#34; nature of laser-induced damage46,47,48,49. We found that these apparent discrepancies could now be explained by differential PARP signaling which gauges the amount and complexity of induced damage32. We confirmed that: 1) laser-microirradiated cells (even after high input-power irradiation) are arrested in interphase in a damage checkpoint control-dependent manner and remain viable (at least up to 48 h)32,50; and 2) repair factor recruitment/modifications faithfully recapitulate those observed with treatment with conventional DNA damaging agents and DSB induction by endonucleases32,39,42,44,50,51,52. These results strongly support the physiological relevance of studying laser damage-induced cellular responses.

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			<td height="42" style="height:42px;width:216px;">Laser Power and Energy Meter (RoHS)</td>
			<td style="width:251px;">Coherent</td>
			<td style="width:264px;">FieldMaxII-TOP&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">ORCA-R2 Digital CCD Camera</td>
			<td style="width:251px;">Hamamatsu</td>
			<td style="width:264px;">C10600</td>
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			<td height="63" style="height:63px;width:216px;">LSM 510 META Laser Scanning Microscopes</td>
			<td style="width:251px;">Carl Zeiss</td>
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			<td height="21" style="height:21px;width:216px;">35mm Gridded Dishes&#160;</td>
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			<td style="width:264px;">P35G-1.5-14-CGRD-D</td>
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			<td height="21" style="height:21px;width:216px;">PtK2 kidney epithelial cells</td>
			<td style="width:251px;">ATCC</td>
			<td style="width:264px;">CCL 56&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HeLa cells</td>
			<td style="width:251px;">ATCC</td>
			<td style="width:264px;">CCL-2</td>
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			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:251px;">Life Technologies</td>
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			<td height="21" style="height:21px;width:216px;">CO2-Independent Medium&#160;</td>
			<td style="width:251px;">Life Technologies</td>
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			<td height="21" style="height:21px;width:216px;">Advanced MEM&#160;</td>
			<td style="width:251px;">Life Technologies</td>
			<td style="width:264px;">12492-013</td>
			<td>supplemented with L-Glutamine, 4% FBS</td>
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			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
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			<td height="21" style="height:21px;">L-Glutamine</td>
			<td style="width:251px;">Fisher Scientific</td>
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			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:251px;">Omega Scientific</td>
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			<td height="21" style="height:21px;width:216px;">Thymidine&#160;</td>
			<td style="width:251px;">&#160;SIGMA</td>
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			<td height="42" style="height:42px;width:216px;">serum free media&#160;&#160;&#160;&#160; (Opti-MEM I Reduced Serum Media)</td>
			<td style="width:251px;">Fisher Scientific</td>
			<td style="width:264px;">11058021</td>
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			<td height="42" style="height:42px;width:216px;">Anti-Cycolbutance pyrimidine dimer (CPD) (mouse)</td>
			<td style="width:251px;">Kamiya Biomedical Company</td>
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			<td height="21" style="height:21px;width:216px;">Anti-53BP1 (rabbit)</td>
			<td style="width:251px;">Santa Cruz Biotech</td>
			<td style="width:264px;">sc-22760</td>
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			<td height="21" style="height:21px;width:216px;">Anti-CtIP (rabbit)</td>
			<td style="width:251px;">Abcam</td>
			<td style="width:264px;">ab70163</td>
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			<td height="21" style="height:21px;width:216px;">Anti-PAR polymers (mouse)</td>
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			<td style="width:264px;">BML-SA216-0100</td>
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			<td height="21" style="height:21px;width:216px;">Anti-PAR (rabbit)</td>
			<td style="width:251px;">Trevigen</td>
			<td style="width:264px;">4336-BPC-100</td>
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			<td height="21" style="height:21px;width:216px;">Anti-TRF2 (mouse)</td>
			<td style="width:251px;">Novus Biological&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Anti 6-4PP (mouse)</td>
			<td>Kamiya Biomedical</td>
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			<td height="42" style="height:42px;width:216px;">Anti-Rad21 (Rabbit)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (for cohesin detection)</td>
			<td style="width:251px;">generated in Yokomori (KY) lab</td>
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			<td height="21" style="height:21px;width:216px;">Anti-Rad51 (Rabbit)</td>
			<td style="width:251px;">Santa Cruz Biotechnology</td>
			<td style="width:264px;">SC-8349</td>
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			<td height="21" style="height:21px;width:216px;">Anti-Ku70 (mouse)</td>
			<td>&#160;Novus Biologicals</td>
			<td style="width:264px;">NB100-102</td>
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			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;">ref 43</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#160;Anti-hCAPG (Rabbit)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (for condensin detection)</td>
			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;">ref 42</td>
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			<td height="42" style="height:42px;width:216px;">Cy3 AffiniPure Donkey Anti-Rabbit IgG</td>
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			<td height="42" style="height:42px;width:216px;">Donkey Anti-Mouse Alexa Fluor 488 IgG</td>
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			<td height="42" style="height:42px;width:216px;">Lipofectamine 2000 Transfection Reagent</td>
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			<td height="21" style="height:21px;width:216px;">GFP-SMC1 stable cell line&#160;&#160;&#160;&#160;</td>
			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;">ref 49&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-NEIL2 stable cell line</td>
			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;">ref 54</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-NTH1 stable cell line</td>
			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;">ref 32</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-SMC1 plasmid</td>
			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-Ku plasmid</td>
			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;"></td>
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			<td style="width:264px;">NB100&#8211;395</td>
			<td style="width:441px;"></td>
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			<td style="width:441px;"></td>
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			<td style="width:251px;">generated in Berns lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;">ref 32</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TRF2-YFP PtK2 stable cell line</td>
			<td style="width:251px;">generated in Berns lab</td>
			<td style="width:264px;"></td>
			<td>ref 32</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
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			<td>4680-096-03</td>
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			<td>N1537</td>
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			<td>118500</td>
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			<td height="21" style="height:21px;width:216px;">Olaparib&#160;</td>
			<td style="width:251px;">Apexbio Technology</td>
			<td>A4154</td>
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			<td>ELECTRON MICROSCOPY SCIENC MS</td>
			<td style="width:264px;">100503-916 (EA)</td>
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			<td style="width:251px;">Bio-Rad</td>
			<td style="width:264px;">SOFT-LIT-70-9600-Q1-469PC&#160;</td>
			<td>image analysis program</td>
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			<td height="21" style="height:21px;width:216px;">Excel</td>
			<td style="width:251px;">microsoft</td>
			<td style="width:264px;"></td>
			<td>spreadsheet program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X100</td>
			<td style="width:251px;">Fisher Scientific</td>
			<td style="width:264px;">BP151-500</td>
			<td>detergent</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s phosphate-buffered saline (DPBS) 10X without calcium and magnesium</td>
			<td style="width:251px;">Fisher Scientific</td>
			<td style="width:264px;">14200166</td>
			<td>dilute to 1X&#160;</td>
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	</tbody>
</table>

laser microirradiation to study in vivo cellular responses to simple and complex dna damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Using PtK2 cells stably expressing EGFP-53BP11220-1711 or TRF2-YFP, laser input power titration was carried out to determine the optimal condition for their recruitment (Figure 1)32.
At high input-power laser damage sites, the significant clustering of CPD (crosslinking damage typically generated by ultraviolet (UV) damage) as well as GFP-NTH1 DNA glycosylase that specifically recognizes base damage were observed. In addition, hi...

Watch this Scientific Journal Video about Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage at JoVE.com

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

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

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

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

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.

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Research

JoVE Journal

Genetics

10.1K visualizações.  University of California, Irvine. 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 ...