This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging (Z01 AG000746-08) and the Fanconi Anemia Research Fund.

1. Preparation of Dig-TMP
<ol>
	<li>Mix 50 mg (0.18 mmoles) of 4&#39;-chloromethyl-4,5&#39;,8-trimethylpsoralen and 590 mg (2.7 mmoles) of 4,7,10-trioxa-1,13-tridecanedi-amine in a dry 25 mL round-bottom flask under nitrogen. Add 10 mL toluene and reflux for 12 h. Remove solvent in a rotary evaporator under reduced pressure.
	<ol>
		<li>Purify the residue by flash column chromatography over silica gel. Elute the column with chloroform, methanol, and 28% ammonia solution (9:1:0.5). Evaporate the solvent...

<ol>
	<li>Lindahl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Intrinsic+Fragility+of+DNA+(Nobel+Lecture).">The Intrinsic Fragility of DNA (Nobel Lecture).</a> Angew. Chem. Int. Ed Engl. 55 (30), 8528-8534 (2016).</li><li>Sirbu, B. M., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+response:+three+levels+of+DNA+repair+regulation.">DNA damage response: three levels of DNA repair regulation.</a> Cold Spring Harb. Perspect. Biol. 5, a01272 (2013).</li><li>Berkovich, E., Monnat, R. J., Kastan, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+protein+dynamics+and+DNA+repair+following+generation+of+DNA+double-strand+breaks+at+defined+genomic+sites.">Assessment of protein dynamics and DNA repair following generation of DNA double-strand breaks at defined genomic sites.</a> Nat. Protoc. 3 (5), 915-922 (2008).</li><li>Rogakou, E. P., Boon, C., Redon, C., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megabase+chromatin+domains+involved+in+DNA+double-strand+breaks+in+vivo.">Megabase chromatin domains involved in DNA double-strand breaks in vivo.</a> J. Cell Biol. 146, 905-916 (1999).</li><li>Lan, L., 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=In+situ+analysis+of+repair+processes+for+oxidative+DNA+damage+in+mammalian+cells.">In situ analysis of repair processes for oxidative DNA damage in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 13738-13743 (2004).</li><li>Reynolds, P., Botchway, S. W., Parker, A. W., O'Neill, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+dynamics+of+DNA+repair+proteins+following+laser+microbeam+induced+DNA+damage+-+when+is+a+DSB+not+a+DSB?.">Spatiotemporal dynamics of DNA repair proteins following laser microbeam induced DNA damage - when is a DSB not a DSB?.</a> Mutat. Res. 756, 14-20 (2013).</li><li>Vilenchik, M. M., Knudson, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+DNA+double-strand+breaks:+production,+fidelity+of+repair,+and+induction+of+cancer.">Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 12871-12876 (2003).</li><li>Vilenchik, M. M., Knudson, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+dose-rate+effects,+endogenous+DNA+damage,+and+signaling+resonance.">Radiation dose-rate effects, endogenous DNA damage, and signaling resonance.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 17874-17879 (2006).</li><li>Aguilera, A., Garcia-Muse, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+of+genome+instability.">Causes of genome instability.</a> Annu. Rev. Genet. 47, 1-32 (2013).</li><li>Swenberg, J. 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=Endogenous+versus+exogenous+DNA+adducts:+their+role+in+carcinogenesis,+epidemiology,+and+risk+assessment.">Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment.</a> Toxicol. Sci. 120 (1), S130-S145 (2011).</li><li>Eichman, B. F., Mooers, B. H., Alberti, M., Hearst, J. E., Ho, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structures+of+psoralen+cross-linked+DNAs:+drug-dependent+formation+of+holliday+junctions.">The crystal structures of psoralen cross-linked DNAs: drug-dependent formation of holliday junctions.</a> J. Mol. Biol. 308, 15-26 (2001).</li><li>Lai, C., 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=Quantitative+analysis+of+DNA+interstrand+cross-links+and+monoadducts+formed+in+human+cells+induced+by+psoralens+and+UVA+irradiation.">Quantitative analysis of DNA interstrand cross-links and monoadducts formed in human cells induced by psoralens and UVA irradiation.</a> Anal. Chem. 80, 8790-8798 (2008).</li><li>Thazhathveetil, A. K., Liu, S. T., Indig, F. E., Seidman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoralen+conjugates+for+visualization+of+genomic+interstrand+cross-links+localized+by+laser+photoactivation.">Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation.</a> Bioconjug. Chem. 18, 431-437 (2007).</li><li>Muniandy, P. A., Thapa, D., Thazhathveetil, A. K., Liu, S. T., Seidman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+laser-localized+DNA+interstrand+cross-links+in+G1+phase+mammalian+cells.">Repair of laser-localized DNA interstrand cross-links in G1 phase mammalian cells.</a> J Biol. Chem. 284 (41), 27908-27917 (2009).</li><li>Nowsheen, S., 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=Accumulation+of+oxidatively+induced+clustered+DNA+lesions+in+human+tumor+tissues.">Accumulation of oxidatively induced clustered DNA lesions in human tumor tissues.</a> Mutat. Res. 674, 131-136 (2009).</li><li>Meyer, B., 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=Clustered+DNA+damage+induces+pan-nuclear+H2AX+phosphorylation+mediated+by+ATM+and+DNA-PK.">Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA-PK.</a> Nucl Acids Res. 41, 6109-6118 (2013).</li><li>Schwab, R. A., Niedzwiedz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+DNA+replication+in+the+vertebrate+model+system+DT40+using+the+DNA+fiber+technique.">Visualization of DNA replication in the vertebrate model system DT40 using the DNA fiber technique.</a> J Vis. Exp. (56), e3255 (2011).</li><li>Kad, N. M., Wang, H., Kennedy, G. G., Warshaw, D. M., Van, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+dynamic+DNA+scanning+by+nucleotide+excision+repair+proteins+investigated+by+single-+molecule+imaging+of+quantum-dot-labeled+proteins.">Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single- molecule imaging of quantum-dot-labeled proteins.</a> Mol. Cell. 37 (5), 702-713 (2010).</li><li>Spielmann, H. P., Sastry, S. S., Hearst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+large-scale+synthesis+of+psoralen+furan-side+monoadducts+and+diadducts.">Methods for the large-scale synthesis of psoralen furan-side monoadducts and diadducts.</a> Proc. Natl. Acad. Sci. U.S.A. 89 (10), 4514-4518 (1992).</li><li>Huang, J., 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=The+DNA+translocase+FANCM/MHF+promotes+replication+traverse+of+DNA+interstrand+crosslinks.">The DNA translocase FANCM/MHF promotes replication traverse of DNA interstrand crosslinks.</a> Mol. Cell. 52, 434-446 (2013).</li><li>Huang, J., 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=Single+Molecule+Analysis+of+Laser+Localized+Interstrand+Crosslinks.">Single Molecule Analysis of Laser Localized Interstrand Crosslinks.</a> Front Genet. 7, 84 (2016).</li><li>Ciccia, A., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+damage+response:+making+it+safe+to+play+with+knives.">The DNA damage response: making it safe to play with knives.</a> Mol. Cell. 40, 179-204 (2010).</li><li>Mori, T., Matsunaga, T., Hirose, T., Nikaido, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+monoclonal+antibody+recognizing+ultraviolet+light-induced+(6-4)+photoproducts.">Establishment of a monoclonal antibody recognizing ultraviolet light-induced (6-4) photoproducts.</a> Mutat. Res. 194, 263-270 (1988).</li><li>Poirier, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisera+specific+for+carcinogen-DNA+adducts+and+carcinogen-modified+DNA:+applications+for+detection+of+xenobiotics+in+biological+samples.">Antisera specific for carcinogen-DNA adducts and carcinogen-modified DNA: applications for detection of xenobiotics in biological samples.</a> Mutat. Res. 288, 31-38 (1993).</li><li>Henderson, 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=Detection+of+G-quadruplex+DNA+in+mammalian+cells.">Detection of G-quadruplex DNA in mammalian cells.</a> Nucleic Acids Res. 42, 860-869 (2014).</li></ol>

The laser localization technology requires the use of adherent cells with nuclei that are visible in bright field microscopy. We have tried to attach nonadherent cells, such as primary lymphocytes, or loosely adherent cultured cells such as AD293, to the glass surface with cell adhesive preparations such as polylysine or collagen, or more complex mixtures. Although these treatments may bind the cells to the surface, we find that they generally stay rounded making it very difficult to focus into the nuclei. Furthermore, t...

DNA is under constant assault from exogenous agents such as radiation, ultraviolet light, environmental toxins, combustion products, etc. Additionally, it is also attacked by endogenous radical species produced by oxidative metabolism. All of these have the potential to chemically or physically disrupt the integrity of DNA 1. Perturbations in the genome can activate the DNA Damage Response (DDR), a recruitment and post translational modification cascade with hundreds, if not thousands, of proteins and microRNAs involved in lesion repair, regulation of the cell cycle, apoptosis, senescence, and inflammatory pathways 2.
Most of our information about the DDR comes from studies with DSBs. This is in large part due to the availability of technologies for introducing breaks, including sequence specific breaks, in genomic DNA in living cells 3. In addition, the propensity of breaks to induce foci of DDR proteins, which can be displayed by immunofluorescence, has been very helpful for identifying the kinetics and requirements of responding proteins. One of the key technologies for studying the DDR was introduced by Bonner and colleagues, who used a laser beam to direct a stripe of DSBs in a &#34;Region of Interest&#34; (ROI) in the nuclei of living cells 4. In effect, they created a lengthy focus in which proteins of the DDR could be identified by immunofluorescence. This was illustrated by their demonstration of the strong stripe of phosphorylated histone H2AX (&#947;-H2AX) in the laser exposed cells. Since then, the laser approach has been employed in numerous studies of the DDR induced by DSBs. Although powerful and popular, and the source of dramatic immunofluorescence images, it should be noted that in most experiments the laser intensity is adjusted so as to produce observable results, without concern for lesion identity, density, or spacing. Indeed, it can be difficult to make these estimates. Thus they are largely ignored, despite the multiplicity of lesions introduced into DNA by lasers 5. This contributes to the many contradictions in the literature 6.
In contrast to DSBs, most chemical modifications of DNA do not stimulate the formation of discrete foci of DDR proteins. This is important in the light of our current understanding of lesion frequencies. It has been estimated that human cells in culture incur as many as 50 DSBs per cell cycle, formed largely during S phase 7,8,9. Fewer are formed in non-proliferating cells. This contrasts with the number of nucleobase losses or modification events, which are in the tens of thousands per cell/day 1,10. Thus, we know most about the DDR induced by events that are relatively rare, and much less about those induced by helix distorting lesions, which in aggregate are far more common.
In order to address questions about the cellular response to covalent modifications of genomic DNA, we wanted to work with a helix distorting DNA adduct that had inherent DDR induction activity. Furthermore, to facilitate experimental design and interpretation we were interested in a structure whose introduction could be controlled with respect to time and was amenable to visualization. Accordingly, we developed a strategy based on psoralen. Psoralens are well characterized photoactive DNA intercalators favoring 5&#39; TA:AT sites. Unlike other crosslinking agents such as nitrogen mustards and mitomycin C (MMC) they are not DNA reactive unless exposed to long wave UV (UVA) light. The intercalated molecules react with thymine bases on opposite strands to produce helix distorting interstrand crosslinks (ICLs) 11. With the trimethyl psoralen used in our experiments most products are ICLs, relatively few monoadducts are generated (less than 10%) 12, and intrastrand crosslinks between adjacent bases on one strand are not formed. Because they are powerful blocks to replication and transcription, psoralen and other crosslinking agents, like cis-platinum and MMC, are commonly used in chemotherapy. Thus psoralen enabled studies that followed the activation of the DDR by a helix distorting structure, and also provided insight into the cellular response to a compound with clinical importance.
We synthesized a reagent in which trimethyl psoralen was linked to digoxigenin (Dig), a plant sterol not found in mammalian cells and frequently used as an immunotag. The requirement for photoactivation permits localization by laser light (365 nm) of psoralen ICLs in defined ROI in nuclei in living cells. These can be displayed by immunofluorescence against the Dig tag. DNA repair and DDR proteins appeared in the stripes of laser localized ICLs 13,14.
The DDR activated by the high laser intensities used to produce DSBs could be due to isolated or clustered damage 15,16. Consequently, the relevance of results from these experiments to naturally occurring lesions, present at much lower concentration, is uncertain. To address similar questions about psoralen adduct frequency and spacing in DNA, we took advantage of DNA fiber technology 17 and immunoquantum dots. Quantum dots are much brighter than fluorescent dyes and are not bleached by exposure to light. Thus they are frequently used for single molecule imaging 18, an application for which fluorescent dyes are insufficiently bright. Individual DNA fibers can be stretched on glass slides and can be displayed by immunofluorescence against nucleoside analogues incorporated during incubations prior to cell harvest. We treated cells with Dig-psoralen and exposed the ROI to laser micro irradiation. Fibers were prepared from the cells and individual Dig-psoralen adducts could be visualized with the immunoquantum dots. Exposing the cells to nucleoside analogues for relatively short times (20-60 min) permits the display of replication tracts in the vicinity of the laser localized ICLs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digoxigenin NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>11333054001</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloro-psoralen</td>
			<td>Berry and Associates</td>
			<td>PS 5000</td>
			<td></td>
		</tr>
		<tr>
			<td>diaminoglycol</td>
			<td>Sigma-Aldrich</td>
			<td>369519</td>
			<td>4,7,10-Trioxa-1,13-tridecanediamine</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Acros Organics</td>
			<td>423550040</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A4524</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium solution</td>
			<td>Sigma-Aldrich</td>
			<td>5002</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC plates</td>
			<td>Analtech, Inc.</td>
			<td>P02511</td>
			<td></td>
		</tr>
		<tr>
			<td>Flass glass column 24/40, 100ml</td>
			<td>Chemglass Life Sciences</td>
			<td>CG-1196-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon T2000_E2 spinning disk confocal microscope, equipped with automated stage and environmental control chamber and plate holder</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>With Volocity Software</td>
		</tr>
		<tr>
			<td>Micropoint Galvo&#160;</td>
			<td>Andor Technologies</td>
			<td></td>
			<td>with a Nitrogen pulsed laser&#160;</td>
		</tr>
		<tr>
			<td>dye cell</td>
			<td>Andor Technologies</td>
			<td>MP-2250-2-365</td>
			<td></td>
		</tr>
		<tr>
			<td>365 dye</td>
			<td>Andor Technologies</td>
			<td>MP-27-365-DYE</td>
			<td></td>
		</tr>
		<tr>
			<td>IdU&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>17125</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm glass botomm plates 1.5 coverslip, 10mm glass diameter, uncoated</td>
			<td>Matek</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>New Comer Supply</td>
			<td>Part # 5070</td>
			<td>New Silane Slides</td>
		</tr>
		<tr>
			<td>Mouse anti BrdU antibody (IdU)</td>
			<td>BD Biosciences</td>
			<td>347580</td>
			<td>1 in 40</td>
		</tr>
		<tr>
			<td>Rat anti BrdU Antibody (CldU)</td>
			<td>Abcam</td>
			<td>ab6326</td>
			<td>1 in 200&#160;</td>
		</tr>
		<tr>
			<td>Rabbit anti Dig antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>710019</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Q-dot 655 goat anti Rabbit IgG</td>
			<td>ThermoFisher Scientific</td>
			<td>Q-11421MP</td>
			<td>1 in 5000</td>
		</tr>
		<tr>
			<td>AF647- goat anti Rat IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>112-605-167</td>
			<td>1 in 100</td>
		</tr>
		<tr>
			<td>AF488-goat anti mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-545-166</td>
			<td>1 in 100</td>
		</tr>
		<tr>
			<td>Zeiss epifluorescent microscope A200</td>
			<td>Zeiss</td>
			<td></td>
			<td>&#160;with Axiovision software</td>
		</tr>
		<tr>
			<td>Q-dot 655 filter</td>
			<td>Chroma</td>
			<td>39107</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Laser localized Dig-TMP (Figure 1A) ICLs can be displayed by immunofluorescence against the Dig-tag linked to the psoralen. Although the laser can be directed to strike in an area of any contour, stripes are not &#34;natural&#34; shapes in cells, and legitimate signals can be easily distinguished from artifacts due to non-specific binding by primary or secondary antibodies. This feature is helpful when using antibodies of less than perfect specificity. An example of the w...

Watch this Scientific Journal Video about Single Molecule Analysis of Laser Localized Psoralen Adducts at JoVE.com

Single Molecule Analysis of Laser Localized Psoralen Adducts

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.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

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

single-molecule-analysis-of-laser-localized-psoralen-adducts

Research

JoVE Journal

Genetics

6.6K visualizações.  National Institute on Aging, National Institutes of Health. The overall goal of this procedure is to perform single molecule analysis of the distribution of laser localized interstrand crosslinks in genomic DNA in order to study cellular responses to DNA interstrand crosslinks. This method can help answer key questions in the DNA repair field, regarding the distribution of DNA lesions introduced by laser technology, a widely used experimental tool. The main advantage of this technique is that it allows direct imaging of DNA lesions introduced by the l...