The authors would like to thank Dr. Samuel H. Wilson at the National Institute of Environmental Health Sciences for the cell lines used in this work.

The authors have nothing to disclose.

Using the conditions described here, any confocal microscope with a UV-A or 405 nm laser, either integrated by the manufacturer or added by the end user, can induce DNA damage within the nucleus of a cell and allow the recruitment of DNA repair proteins to the site of induced DNA damage to be monitored. However, as noted in the protocol and representative results, wavelength selection, applied power, and damage characterization are all important issues that must be addressed first by the user in order to study a specific DNA repair pathway. Additionally, there are other considerations in experimental design that must also be considered by users.
In order to monitor a protein&#39;s behavior in live cells post micro-irradiation, a fluorescently labeled protein must be created. The availability of a wide range of fluorescent proteins that can be spectrally separated offers tools for creating protein fusions that can be used for characterizing cellular responses to the induction of DNA damage. Transient transfection of fluorescent proteins of interest allows for quick screening of damaging conditions, mutations, or even inhibitors, while stably transfected cells offer more control over expression levels and allow for other biochemical characterizations to be performed, validating micro-irradiation results. Spectrally distinct fluorescent proteins can also be utilized in the same cell to monitor interactions between proteins within the same pathway or in different pathways. Fluorescent proteins also eliminate the need for specific antibodies for each protein of interest and allow live cell monitoring of protein behavior for long periods of time after the induction of damage.
However, the use of fluorescent proteins also has drawbacks. Without genetic backgrounds deficient in the protein(s) of interest, endogenous proteins will compete with the fluorescently tagged proteins. Conjugation of the fluorescent tag to the N or C terminus of the protein may alter protein folding, hinder key protein-protein interactions, or block translocation signals; altering function and potentially impacting recruitment dynamics. These effects have been demonstrated in a number of reports where immunofluorescent staining demonstrated dramatically different recruitment and retention times for endogenous proteins at the damage site28. Use of transient transfection or stable clones can also alter the response observed, typically through variations in protein expression levels. Further, the use of multiple fluorescent proteins in a single experiment may also change the dynamics of recruitment, if protein levels are not equilibrated well or if recruitment of the larger fluorescently-tagged protein blocks the recruitment of other proteins. Finally, fluorescent proteins can act as weak sensitization agents, increasing the formation of reactive oxygen species, and potentially altering the DNA damage mixtures induced46,47. Despite these drawbacks, fluorescent proteins offer a number of distinct advantages in studying DNA repair with micro-irradiation, and if users incorporate appropriate controls, such as the damage characterization described here, they can provide new insight into damage response and protein-protein interactions3.
If live cell imaging is not required, immunofluorescence can be used to monitor the response to induced damage. By fixing cells at specific times post damage induction and staining with antibodies specific for proteins and lesions of interest, static snapshots of damage induction and the recruitment and retention of repair proteins can be built. Antibodies can be used to monitor the recruitment of multiple proteins of interest and/or post-translational modifications induced by the DNA damage response. Use of immunofluorescence eliminates the need for fluorescent proteins, and allows for endogenous protein behavior to be examined. However, this method too has its drawbacks. Highly specific antibodies are necessary, and permeabilization and blocking procedures need to be optimized to allow for detection of recruitment with sufficient signal intensity. Fixing procedures are not instantaneous, and this physical constraint limits the temporal resolution of this approach. Mapping the site of damage induction so cells can be re-located after staining can also present significant challenges. The confocal microscope used in this work allows for image-based registration as described above, so damaged cells can be relocated with high precision. If stage registration or etched coverglass is not available, the time investment involved in relocating cells without stage registration, coupled with the inherent delay between damage induction and the imaged recruitment, may make immunofluorescence unattractive to some users. However, the most accurate and complete micro-irradiation experimental designs will incorporate these types of approaches in parallel with the use of fluorescent proteins, as described in the presented protocol.
Here, both live cell imaging and immunofluorescence is used to demonstrate the utility of laser micro-irradiation. Immunofluorescent staining allows us to accurately examine the mixture of DNA damage created and the recruitment of proteins induced by each laser power, which allow us to better interpret the observed alterations in the recruitment and retention of XRCC1-GFP. Based on these results, the use of 405 nm lasers should be limited for examination of BER and SSBR proteins. Further, the optimal experimental design would include power measurements after the objective, a full damage mixture characterization for each cell line used, and validation of the recruitment and retention observed in fluorescently tagged proteins with immunofluorescence. Obviously, equipment, time, and cost considerations may make these optimal experimental designs impossible for some users. However, the importance of each of these elements is demonstrated here, and users should keep these considerations in mind when beginning micro-irradiation experiments.

Fluorescent microscopy has emerged as a powerful technique to visualize cellular architecture, examine protein localization, and monitor protein-protein and protein-DNA interactions. Using fluorescent microscopy to study DNA damage responses after the application of global DNA damaging agents, such as ultraviolet (UV) light, ionizing radiation, chemical oxidizing or alkylating agents, and/or chemotherapeutics has provided new insight into the initiation, signaling, and recruitment of DNA repair proteins to sites of DNA damage1,2. However, these global and asynchronous damaging events are limiting, if detailed information about the recruitment order, kinetics of association or dissociation, and the relationships between key DNA repair proteins are sought. Fortunately, advancements in laser scanning confocal microscopes, broader availability of damage-inducing laser wavelengths, and improvements in fluorescent proteins over the past 25 years have provided researchers with improved tools for examining these aspects of DNA repair, through targeted DNA damage induction.
Irradiation of cells with laser microbeams in order to study cellular and subcellular functions is a well-established tool in cell and radiation biology3. Application of this technique to the study of DNA repair emerged when Cremer and co-workers used a highly-focused UV (257 nm) laser microbeam system to induce DNA damage over a 0.5 &#181;m spot in Chinese hamster ovary (CHO) cells4 and established the induction of DNA photolesions by this system5. While offering significant improvements over UV microbeam methods at the time, adoption of this damaging system was limited due to its specialized set up, and its inability to generate double strand breaks (DSBs)6. Subsequent investigation of a variety of UV-B (290 - 320 nm) and UV-A (320 - 400 nm) wavelengths by a number of groups revealed that UV photoproducts, oxidative base lesions, single strand breaks (SSBs), and DSBs could be induced dependent on the laser wavelength and power applied4,7,8,9,10 (reviewed in 3). Further, combinations of these UV-B and UV-A wavelengths with sensitizing agents, like psoralen, bromodeoxyuridine (BrdU), and Hoechst dyes, were also found to induce DNA damage dependent on wavelength, power, and duration of exposure, though the power needed to induce damage is often lowered in the presence of these agents11,12,13,14,15. These advancements expanded the use of micro-irradiation, though there were still technical hurdles to be addressed for wider adoption of these methods.
Cremer and co-workers significantly advanced the field of micro-irradiation by precisely focusing the UV microbeam to apply significant damaging energy over a highly localized area in the cell. As confocal microscopes and laser microdissection systems advanced, tightly focused light was more broadly accessible; however, coupling UV sources to scopes and dealing with the chromatic aberrations they induced still presented significant challenges to most users3,6,16. As UV dyes increased in popularity throughout the 1990s, optics capable of focusing and capturing UV-excited fluorescence became more widely available16, and improvements in laser scanning offered users the ability to create highly focused UV excitation spots within cells6,17. However, it was not until the early 2000s that the true impact of this combination of tightly focused beams with higher intensity lasers was felt, when numerous reports emerged demonstrating that DNA strand breaks could be induced with and without sensitizers in the UV-A range6,10,18,19,20, 405 nm21,22,23,24,25,26, and even at longer visible wavelengths like 488 nm27. These advancements allowed for more widespread adoption of the micro-irradiation technique on a number of commercial systems. In parallel with these developments, two-photon techniques also emerged that allowed precise induction of DNA damage; though these advancements will not be discussed here, there are a number of review articles discussing these methodologies9,28,29,30.
With the current accessibility of confocal microscopes capable of delivering highly focused UV light and the wide-spread availability of fluorescent proteins to allow real-time tracking of DNA repair proteins, micro-irradiation techniques have evolved into powerful tools for examining DNA damage response and repair pathways. However, users need to be aware that the generation of DNA damage is highly dependent on the laser wavelength and the power applied to the sub-nuclear region. Use of UV-C (~260 nm) wavelengths allow direct DNA excitation and high selectivity for the induction of UV photoproducts7,8. UV-B and UV-A wavelengths produce mixtures of DNA damage (base lesion, SSBs, and DSBs), dependent on the applied power and the cellular background utilized7. Endogenous photosensitizers and anti-oxidant levels in the targeted cells can influence the DNA damage mixtures produced by these wavelengths. Additionally, the use of exogenous photosensitizers (BrdU, etc.) may assist in lowering the energy needed for the induction of DNA damage. However, these agents can induce DNA damage by themselves, and they may alter the cell cycle and chromatin structure, so their use may produce undesired effects that need to be considered by the user. Therefore, prior to using micro-irradiation to study DNA damage response and repair, careful consideration of the DNA repair pathway of interest, the wavelengths available for use, and the DNA damage mixture created is required.
Here, laser micro-irradiation is performed at two commonly used wavelengths, 355 nm and 405 nm, without sensitizers to demonstrate the mixtures of DNA damage induced by these wavelengths and the influences these damage mixtures have on examining the repair of SSBs and DSBs. Users should be aware that these wavelengths do not create a single species of strand breaks or base lesions. In order to discriminate between DNA repair pathways, users must carefully control the applied power over a specific region of the nucleus and characterize the induced damage using multiple strand break markers and DNA lesion antibodies. If properly applied and characterized, laser micro-irradiation can enrich some species of DNA damage, allowing users to assess repair of base lesions and SSBs or DSBs, with some specificity. Therefore, we have provided a method allowing users to reproducibly perform laser micro-irradiation, characterize the DNA damage mixtures induced by the applied laser dose, and perform data analysis.

<table style="border-collapse:collapse;width:548pt" width="730">
	<colgroup>
		<col style="width:220pt" width="293" />
		<col style="width:119pt" width="158" />
		<col style="width:84pt" width="112" />
		<col style="width:125pt" width="167" />
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	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl23" height="21" style="width:220pt;height:15.75pt" width="293">Nikon A1rsi laser scanning confocal microscope</td>
			<td class="xl21" style="width:119pt" width="158">Nikon</td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23" style="width:125pt" width="167"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl23" height="21" style="height:15.75pt">NIS Elements software</td>
			<td class="xl21" style="width:119pt" width="158">Nikon</td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">355 nm laser</td>
			<td class="xl23">PicoQuant VisUV</td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23">Radiation source</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Galvanometer photoactivation miniscanner</td>
			<td class="xl21" style="width:119pt" width="158">Bruker</td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Microscope slide photodiode power sensor</td>
			<td class="xl21" style="width:119pt" width="158">THORLabs</td>
			<td class="xl21" style="width:84pt" width="112">S170C</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Fiber photodiode power sensor</td>
			<td class="xl21" style="width:119pt" width="158">THORLabs</td>
			<td class="xl21" style="width:84pt" width="112">S150C</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Digital handheld optical power and energy meter</td>
			<td class="xl21" style="width:119pt" width="158">THORLabs</td>
			<td class="xl21" style="width:84pt" width="112">PM100D</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">CHO-K1</td>
			<td class="xl21" style="width:119pt" width="158"></td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23">From Dr. Samuel H. Wilson, NIEHS</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Minimal essential media</td>
			<td class="xl28">Hyclone</td>
			<td class="xl21" style="width:84pt" width="112">SH3026501</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Fetal bovine serum</td>
			<td class="xl21" style="width:119pt" width="158">Atlanta biologicals</td>
			<td class="xl21" style="width:84pt" width="112">S11550</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">XRCC1-GFP</td>
			<td class="xl21" style="width:119pt" width="158">Origene</td>
			<td class="xl27">RG204952</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Jetprime</td>
			<td class="xl21" style="width:119pt" width="158">Polyplus transfection</td>
			<td class="xl27">11407</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Geneticin</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl27">10131035</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">4 chambered coverglass</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">155382</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">8 chambered coverglass</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">155409</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Anti 53BP-1</td>
			<td class="xl21" style="width:119pt" width="158">Novus</td>
			<td class="xl21" style="width:84pt" width="112">NB100304</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Anti-phospho-histone H2AX&#160;</td>
			<td class="xl21" style="width:119pt" width="158">Millipore</td>
			<td class="xl21" style="width:84pt" width="112">05-636-I</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Anti cyclobutane pyrimidine dimer</td>
			<td class="xl21" style="width:119pt" width="158">Cosmo Bio clone</td>
			<td class="xl21" style="width:84pt" width="112">CAC-NM-DND-001</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Anti 8-oxo-2&#180;-deoxyguanosine&#160;</td>
			<td class="xl21" style="width:119pt" width="158">Trevigen</td>
			<td class="xl21" style="width:84pt" width="112">4354-MC-050</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Alexa 488 goat anti-mouse</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">A11029</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Alexa 546 goat anti-rabbit</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">A11010</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">4&#8217;,6-Diamidino-2-Phenylindole (DAPI)</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">R37606</td>
			<td class="xl23">Caution toxic!</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Phosphate buffered saline</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">0780</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Normal goat serum</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">31873</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Bovine serum albumin (BSA)</td>
			<td class="xl21" style="width:119pt" width="158">Jackson Immuno Research</td>
			<td class="xl21" style="width:84pt" width="112">001-000-162</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">37% Formaldehyde</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">9311</td>
			<td class="xl23">Caution toxic!</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Ethanol</td>
			<td class="xl20">Decon Labs</td>
			<td class="xl20">2716</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Methanol</td>
			<td class="xl21" style="width:119pt" width="158">VWR</td>
			<td class="xl21" style="width:84pt" width="112">BDH1135</td>
			<td class="xl23">Caution toxic!</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">HCl</td>
			<td class="xl20">Fisher</td>
			<td class="xl20">SA49</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Sodium azide</td>
			<td class="xl21" style="width:119pt" width="158">Sigma-Aldrich</td>
			<td class="xl18" style="width:84pt" width="112">S2002</td>
			<td class="xl23">Caution toxic!</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:220pt;height:15.75pt" width="293">Tris Hydrochloride</td>
			<td class="xl20">Amresco</td>
			<td class="xl20">O234</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Triton X-100</td>
			<td class="xl21" style="width:119pt" width="158">Sigma-Aldrich</td>
			<td class="xl21" style="width:84pt" width="112">T8787</td>
			<td class="xl29"></td>
		</tr>
	</tbody>
</table>

application of laser micro-irradiation for examination of single and double strand break repair in mammalian cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Characterization of induced DNA damage 
Induction of base lesions and strand breaks is dependent on the laser dose applied to the selected nuclear area and the cellular microenvironment of the cell model used7. Fluorescent proteins fused to repair proteins, like XRCC1, 53BP1, Ku70, or Rad51, provide useful single and double strand break markers for establishing the minimal energy required to see accumulation of a fluorescent protein within a damage ROI above the background fluorescence9,19,31. Once conditions that induce a response are found, it is critical to characterize the damage mixture induced by that specific wavelength and dose. Attenuation of the dose and duration at the wavelength used can allow the user to minimize the formation of complex damage mixtures. Low laser doses in the UV-A range have been demonstrated to produce predominantly SSBs and a small amount of base lesions, appropriate for studying SSBR and BER pathways10,28. Increasing the dose creates more complex base lesions, oxidative and UV induced, and induces more significant numbers of DSBs7,10. While the induction of a single species of DNA damage is desirable for examination of specific DNA repair pathways, it is more likely that users are inducing a mixture of DNA lesions, with a specific lesion like SSBs being much more frequent than base lesions or DSBs. This is similar to DNA damage mixtures induced by chemical agents like hydrogen peroxide (H2O2) or methyl methanesulfonate (MMS)32. Users need to be aware when they report results that damage mixtures can occur, and careful characterization of the dose and lesions at the induced damage site are necessary to ensure the reproducibility and comparability of their results.
In micro-irradiation studies, XRCC1-GFP is often used as a marker for the induction of base lesions and SSBs9,28. XRCC1 is a scaffold protein that plays an important role in SSB repair (SSBR) and base excision repair (BER), and also participates in other repair pathways, like nucleotide excision repair (NER)33,34,35. It plays an important coordinating role in DNA repair, interacting with a number of key proteins, including poly(ADP-ribose) polymerase 1 (PARP-1), DNA polymerase &#946; (Pol &#946;), and DNA ligase III. We utilized XRCC1-GFP stably expressed in CHO-K1 cells to determine the laser doses required to generate SSBs and DSBs. We first identified the minimum dose required to induce an observable recruitment of XRCC1-GFP for each wavelength (Figure 1). For the 355 nm wavelength, a 2 s dwell time over the defined damage ROI generated an increased fluorescent signal within that ROI, indicating the induction of DNA damage that was detectable over the background (Figure 1A). For 405 nm, an 8 fps scan rate was needed to generate an observable recruitment to the damage ROI (Figure 1A). The dose was then increased (10 s for 355 nm and 0.5 fps for 405 nm) to create a more intense damage ROI (Figure 1A).
Recruitment and retention of XRCC1-GFP at the site of induced damage was then monitored by timelapse imaging. Retention of the protein at the site of DNA damage may indicate on-going DNA repair, while dissociation of the protein from the site of induced damage is often considered a marker for completion of BER or SSBR. However, there has been no clear evidence linking the dissociation of XRCC1 from laser-induced DNA damage sites with the completion of repair. Recruitment of the protein to the site of damage is measured by reporting the mean intensity of the fluorescent signal within the damaged ROI over the mean fluorescent signal measured for the entire nucleus (Figure 1B). This type of normalization helps address intensity fluctuations in the nuclear signal, though other normalization techniques can be employed depending on the cellular distribution of the protein of interest. Here, XRCC1 is localized in the nuclear compartment, so normalization to the nuclear area measures the redistribution of the signal to the damage ROI. The ROI mean fluorescent intensity is then recorded for each image in the timelapse, including the pre-damage image, and graphed as a function of time (Figure 1C).
We then further characterized the damage induced by the two selected laser doses to examine the formation of DNA base lesions. First, formation of CPD, a bulky UV-induced lesion, was probed by immunofluorescence as a marker for NER type lesions (Figure 2A). Then, the oxidatively induced base lesion8-oxodG was probed as a marker for BER type lesions (Figure 2B). No significant increase in CPD lesions were observed at the low dose exposure for both wavelengths (2 s for 355 nm and 8 fps for 405 nm), while the high dose treatment at both wavelengths (10 s for 355 nm and 0.5 fps for 405 nm) did show a significant increase in fluorescent signal observed within the damage ROI (Figure 2A). A scatter plot of the CPD ROI mean intensity for each damaged cell shows heterogeneity in damage formation and detection at both wavelengths and doses, indicating that a low level of CPD lesions may be present at the lower doses, but the load may not be significantly detected until a higher dose is applied. The scatterplot also suggests that inefficiency in antibody detection that may limit accurate quantification of the damage mixtures.
This is further highlighted in the detection of oxidatively induced DNA lesions by the marker 8-oxodG. No clear increase in fluorescent signal within the damage ROI was observed for 8-oxodG at either laser wavelength or dose used (Figure 2B). The antibody used for this work is consistent with previous publications9,10,36; however, it should be noted that there can be limitations in observing the formation of 8-oxodG with antibodies37,38. Confirmation of the lack of oxidatively induced lesions is also recommended by a second marker, like recruitment of 8-Oxoguanine DNA Glycosylase (OGG1), the enzyme responsible for removal of 8-oxodG from the DNA10. We did not observe OGG1 recruitment to our DNA damage sites; however, the formation of low levels of oxidatively induced DNA damage cannot be ruled out completely.
Finally, we examined the formation of DSBs using two markers, &#947;H2AX and 53BP-1, at the selected laser doses by immunofluorescence (Figure 3 &#38; 4). &#947;H2AX is commonly used as a strand break marker, but its specificity for DSBs has been questioned in a number of reports39,40. Additionally, it is a phosphorylation event that propagates from the strand break site, so localization of the signal to a strand break can be limited due to this signal propagation. Therefore, combining &#947;H2AX with 53BP-1 allows for more accurate assessment of DSB formation within the damage ROI.
The response of &#947;H2AX and 53BP-1 to micro-irradiation is both wavelength and dose dependent. The low dose (2 s) stimulation at 355 nm elicits no response at 5 and 20 min, and a weak and variable response 10 min post irradiation (Figure 3A) for both markers. The high dose (10 s) of 355 nm micro-irradiation induces an increased fluorescent signal within the damage ROI at 5, 10, and 20 min post irradiation that is reduced at 40 min (Figure 3B). These results indicate that careful titration of the 355 nm dose is required to minimize cross-stimulation of repair pathways, as demonstrated by the reduced detection of the double strand break marker &#947;H2AX at the early time points (&#60; 20 min) at the low dose applied.
Similar experiments were performed using both low (8 fps) and high dose (0.5 fps) 405 nm laser stimulation (Figure 4). At this wavelength significant accumulation of fluorescent intensity within the damage ROI was observed for both 53BP-1 and &#947;H2AX regardless of applied dose, indicating that these doses generate a complex mixture of single and double strand breaks almost immediate after the induction of DNA damage (Figure 4). Additionally, the high doses of 405 nm show an increase in pan-nuclear &#947;H2AX staining within 10 min of damage induction (Figure 4B, bottom) that makes detection of the damage ROI difficult to report, while the 53BP-1 accumulation is more contained within the damage ROI.
These results clearly demonstrate that 405 nm micro-irradiation is not appropriate for monitoring SSBR or BER, and that multiple markers for DNA adducts and strand breaks should be employed to fully characterize the induced lesions and DNA repair responses.
Dose-dependent alteration in recruitment and retention of XRCC1-GFP 
Once the induced DNA damage has been characterized, laser micro-irradiation can be an ideal platform for studying the dynamics of DNA repair proteins. The retention and dissociation kinetics of the XRCC1-GFP shows a dose dependency (Figure 1), which is not unexpected given the induction of different damage mixtures by each wavelength. The highest irradiation doses (10 s and 0.5 fps) show a higher intensity recruitment of XRCC1-GFP relative to the lower doses and longer retention of the XRCC1-GFP at the site of damage over the 20 min time course (Figure 1C). This indicates that the DNA damage created at higher doses for both 355 and 405 nm is likely not resolved during the course of the experiment, which is consistent with the appearance and retention of DSB markers, &#947;H2AX and 53BP-1 (Figures 3 &#38; 4).
Interestingly, the lower damage doses (2 s and 8 fps) show rapid recruitment of XRCC1-GFP to the damage sites and dissociation of XRCC1-GFP over the experimental time course to pre-irradiation levels (Figure 1C). Without the complete characterization of the damage mixture, this may lead to the conclusion that SSBs and base lesions are completely resolved using these damaging conditions. However, the presence of &#947;H2AX and 53BP-1 at 40 min for 355 nm and at 5 min for the 405 nm may lead to different interpretations. For the 355 nm 2 s dose, the damage mixture may be predominantly SSBs, so the appearance of DSB markers at 40 min may indicate that some unrepaired lesions may be leading to DSBs or that DSBs generated by this energy are repaired on a longer time scale. Time scale differences between SSBR and DSB repair have been previously reported28,41,42. Similarly, the 405 nm low dose (8 fps) dissociation may indicate a low level of SSBs or a clustering of SSBs that are rapidly converted to DSBs, which has been noted for high damage micro-irradiation and other DNA damaging agents previously43,44,45.
Together these results highlight the importance of characterizing the induced damage mixtures and utilizing multiple DNA repair proteins and markers for interpreting the recruitment and retention of DNA repair proteins at sites of induced damage.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56265/56265fig1.jpg" /> 
Figure 1. Laser micro-irradiation induces recruitment of XRCC1-GFP.&#160; 
(A) CHO-K1 cells stably expressing XRCC1-GFP were irradiated and imaged before and immediately after damage induction. Arrows indicate location of micro-irradiation dose and scale bar is 10 &#181;m. (B) Recruitment is measured by determining the mean fluorescent intensity within the damage ROI and normalizing to the mean fluorescent intensity of the entire nucleus. (C) Dynamics of recruitment can be measured for each timelapse image.Graphs are representative of two independent experiments with error bars representing the SEM (n=24).&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56265/56265fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56265/56265fig2.jpg" /> 
Figure 2. Laser micro-irradiation induces nucleotide damage.&#160; 
CHO-K1 cells were subjected to laser micro-irradiation and fixed immediately after damage induction. Immunofluorescence was performed to detect CPD and 8-oxodG adducts. (A) Top, scatter plot of the ROI mean fluorescence intensities observed in damaged cells after CPD staining. Bottom, representative images of CPD staining. Arrows indicate location of micro-irradiation and the scale bar is 10 &#181;m (n=12). (B) Representative images for 8-oxodG staining. Arrows indicate location of micro-irradiation and the scale bar is 10 &#181;m.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56265/56265fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56265/56265fig3.jpg" /> 
Figure 3. DNA double strand break markers respond to 355 nm micro-irradiation in a dose dependent manner.&#160; 
CHO-K1 cells were subjected to micro-irradiation and fixed at the time points indicated post-stimulation. Immunofluorescence for the DSB markers &#947;H2AX and 53BP-1 was performed. (A) Scatter plot of normalized damage ROI mean fluorescence intensities measured for undamaged and damaged cells. Error bars are representative of the SEM (n=12). (B) Representative images for &#947;H2AX and 53BP-1 staining. Arrows indicate location of micro-irradiation and the scale bar is 10 &#181;m.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56265/56265fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56265/56265fig4.jpg" /> 
Figure 4. DNA double strand break markers robustly respond to 405 nm micro-irradiation.&#160; 
CHO-K1 cells were subjected to micro-irradiation and fixed at the time points indicated post stimulation. Immunofluorescence for the DSB markers &#947;H2AX and 53BP-1. (A) Scatter plot of normalized damage ROI mean fluorescence intensities measured for undamaged and damaged cells. Error bars are representative of the SEM (n=12). (B) Representative images for &#947;H2AX and 53BP-1 staining. Arrows indicate location of micro-irradiation and the scale bar is 10 &#181;m.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56265/56265fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells at JoVE.com

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

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Research

JoVE Journal

Cancer Research

9.6K Views.  University of South Alabama Mitchell Cancer Institute. Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Video: Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

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Genetics

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.

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.

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

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Biology

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
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This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...