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 in a rotary evaporator under reduced pressure, and recover the pure 4&#39;-[N-(13-amino-4,7,10-trioxatrideca)]aminomethyl-4,5&#39;,8-trimethylpsoralen as a pale yellow viscous liquid.</li>
		<li>Mix 5.5 mg (0.012 mmoles) of 4&#39;-[N-(13-amino-4,7,10-trioxatrideca)]aminomethyl-4,5&#39;,8-trimethylpsoralen with 5 mg (0.008 mmoles) of digoxigenin NHS ester in a dry round-bottom flask under nitrogen. Add 0.5 mL anhydrous dimethyl formamide (DMF) and 3.4 &#181;L of trimethylamine and stir at 50 &#176;C for 18 h.</li>
		<li>Remove solvent in a rotary evaporator under reduced pressure.</li>
		<li>Dissolve the residue in minimum amount of dichloromethane. Apply the solution in 2 &#181;L spots horizontally across the bottom of a preparative thin layer plate with silica gel as a stationary phase. Run the preparative TLC in chloroform: methanol: 28% ammonium hydroxide (8:1:0.1).</li>
		<li>Mask the plate except for the vertical edges and identify the band of product with short wavelength UV 254 nm lamp. Scrape the product from the plate with a spatula and isolate the pure product using chloroform: methanol: 28% ammonium hydroxide (8:1:0.1) mixture.</li>
		<li>Remove solvents in a rotary evaporator under reduced pressure. Dissolve the residual pellets in 1 mL of 50% EtOH:H2O, dispense into 50 &#181;L aliquots in 1.5 mL tubes (~20 aliquots) and dry in a centrifugal evaporator until all solvent is evaporated (~3 h).</li>
		<li>Redissolve each aliquot in 200 &#181;L of 50% EtOH:H2O and dry again in a centrifugal evaporator. Dissolve each aliquot again in 200 &#181;L of 50% EtOH:H2O, dry in a centrifugal evaporator and store pellets at -20 &#176;C for long term storage.</li>
	</ol>
	</li>
	<li>For use, dissolve the pellet in 50 &#181;L of 50% EtOH:H2O. Prepare a 1:100 dilution of the dissolved Dig-TMP in H2O and measure OD at 250 nm. The extinction coefficient of Dig-TMP is 25,000. The solution is stable for about a month if stored at -20 &#176;C. Verify the concentration by measuring OD at 250 nm before each use.
	<ol>
		<li>Calculate stock concentration: Abs x 100 x106/25,000 = Concentration (in &#181;M). Typically the stock solution is about 3 mM. It is important to be in this range in order to minimize the volume of EtOH added to the medium.</li>
	</ol>
	</li>
</ol>
2. Laser Localized Dig-TMP ICLs
<ol>
	<li>Perform laser localization with a confocal microscope with a SRS nitrogen laser (337 nm) pumped through a dye cell emitting a line of 365 nm and firing 3 ns pulses at 10 Hz, with a power of 0.7 nW.</li>
	<li>Make a vertical mark on the side of a 35 mm glass bottomed cell culture dish with a marking pen. Scratch a cross in the center of the glass on the culture surface with a diamond pen 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.</li>
	<li>Sterilize the plate after marking with a rinse of 70% EtOH. Dry before plating cells.</li>
	<li>Plate cells (standard laboratory strains such as HeLa or U2OS) in the cross marked culture dishes one or two days before. Cell should be actively dividing and 50-70% confluent on the day of the experiment. Incubate 24 h with 1 &#181;M 5-chloro-2-deoxyuridine (CldU) to uniformly label DNA.</li>
	<li>Add Dig-TMP in 50% EtOH: H2O to cell culture medium to a final concentration of 20 &#181;M. Bring the medium to 37 &#176;C. Change the medium over the cells and place in incubator (37 &#176;C, 5% CO2) for 30 min to allow the Dig-TMP to equilibrate.</li>
	<li>While the cells are incubating, establish the x/y coordinates of the field of view in which the laser is active. Follow the manufacturer&#39;s calibration procedure in which the laser is directed by the software to etch a mirrored glass slide along a horizontal and diagonal line. The two lines define the boundaries of the field in which the laser can strike a target.</li>
	<li>Place the plate in the environmental chamber, at 37 &#176;C with controlled CO2 and humidity, on the microscope stage and focus with the 60X oil objective on the intersection of the cross.</li>
	<li>Direct the 365 nm laser beam to an ROI, established by the investigator with the shape tool in the software, to form a stripe of 3 &#181;m x 0.6 &#181;m. The outline of the ROI is placed by the cursor in nuclei of cells located in the immediate vicinity of the intersection of the cross. Adjust the z focal plane to target the laser midway through the nucleus. Use a laser in the range of 350-370 nm. 405 nm lasers cannot induce crosslinks 19. 
	NOTE: We locate the ROI in nucleoplasmic areas outside the nucleoli, which can be distinguished from nucleoplasm in differential interference contrast (DIC) imaging.</li>
	<li>Verify the focus and activity of the laser by increasing the power setting to a level sufficient to obliterate a cell (the cell will darken and then disappear). This is an important diagnostic for an unimpeded light path for the laser through the microscope and then through the coverslip and into the cells.
	<ol>
		<li>Lower the power setting to just enough to activate the psoralen and the ICL induced DDR (this must be determined empirically for each laser/microscope combination). Use a laser intensity setting (1.7% here) that does not activate the DDR in the absence of psoralen (monitored by the formation of &#947;-H2AX). 
		NOTE: This is an important determination and the settings may require adjustment if the laser source or a component in the light path is changed. Follow the accumulation of GFP tagged repair proteins such as XPC or FAN1, both of which are rapidly recruited (in seconds) to the laser psoralen stripes, but not to ROI exposed to the laser alone. Some proteins of the DDR appear within seconds, while several minutes may be required for others. It is necessary to perform time course determinations for each protein of interest. We also note that in cells that were not exposed to the laser there are no stripes of responding proteins.</li>
	</ol>
	</li>
	<li>After all cells in a field have been targeted move the plate to a new field, staying close to the intersection of the cross.</li>
	<li>In experiments designed to determine the distribution of inter lesion distances expose cells to the laser in 20-25 fields (4-5 cells/field) around the intersection. In order to analyze replication patterns in the vicinity of the localized ICLs incubate cells with 10 &#181;M 5-Iodo-2-deoxyuridine (IdU) after laser firing. 
	NOTE: The time of incubation with IdU is somewhat arbitrary. We have used as short as 20 min and as long as 60 min (see below). Longer pulses (a few hours) often result in multiple replication tracts coalescing, thus losing the opportunity to image the progress of single forks. In some experiments ces are labeled with CldU for 24 h to uniformly label DNA prior to exposure to Dig-TMP followed by pulse labeling of replication tracts.</li>
</ol>
3. Harvest of Cells and Stretching of DNA Fibers
<ol>
	<li>Remove the plate from the stage, remove the medium, and wash with phosphate buffered saline (PBS). Remove the PBS solution. Place a 10 &#181;L drop of a commercial trypsin/EDTA solution on the intersection of the cross in the middle of the glass surface.</li>
	<li>Incubate for 3-4 min at room temperature (RT) and then draw the trypsin solution with the detached cells into the pipet tip. There is no need to neutralize the trypsin.</li>
	<li>Place the drop of trypsin/EDTA with the cells at one end of a silanized glass microscope slide. Lyse the cells and release the DNA by addition of 10 &#181;L of 0.5% SDS solution and incubate for 3-4 min at RT mixing gently with the pipet tip, allowing the periphery of the pool to dry.</li>
	<li>Tilt the slide 20&#186; and allow the liquid to run down to the end. The DNA fibers are extended/stretched by the hydrodynamic forces of the flowing liquid. Let the slides air dry (~10 min) and fix in 3:1 methanol/acetic acid for 10 min. Remove the slides from the fix solution and air dry again. At this point they can be stored indefinitely in 70% EtOH at -20 &#176;C. On removal from the 70 % EtOH allow to air dry before the next step.</li>
	<li>Wash slides in PBS, and then denature in 2.5 M HCl for 1 h at RT. This treatment depurinates DNA making the incorporated halogenated nucleobases accessible to antibodies.</li>
	<li>Neutralize slides in 0.4 M Tris-HCl, pH 7.4. Wash twice in PBS/0.5% Tween-20 (PBST) for 5 min each.</li>
	<li>Block slides with 5% bovine serum albumin (BSA) and 10% goat serum in PBS. Cover the slides gently with a parafilm to spread the blocking solution evenly over the slide and incubate for 1 h at RT.</li>
	<li>Pipet 100 &#181;L of 1:200 dilution in blocking solution of rat anti-bromodeoxyuridine (specifically detects CldU) primary antibody to each slide. Cover the slides gently with a parafilm to spread the dilution evenly over the slide and incubate in a humidified chamber for 1 h at RT. Remove the parafilm and wash the slides three times in PBST for 5 min each.</li>
	<li>Pipet 100 &#181;L of diluted (1:100) goat anti-rat Alexa Fluor 647 in blocking solution to each slide. Cover the slides gently with a parafilm and incubate for 45 min at RT. Wash the slides three times in PBST for 5 min each.</li>
	<li>Pipet 100 &#181;L of diluted (1:40) mouse anti-bromodeoxyuridine (detects ldU and CldU. This dual specificity necessitates the blocking of CldU by the rat anti BrdU in the prior incubation) primary antibody and rabbit anti-DIG antibody (1:200) in blocking solution to each slide. Cover the slides gently with a parafilm and incubate in a humidified chamber for 1 h at RT. Wash the slides three times in PBST for 5 min each.</li>
	<li>Pipet 100 &#181;L of diluted (1:100) goat anti-mouse Alexa Fluor 488 andc(1:5,000) goat anti-rabbit probe (e.g., Qdot 655) in blocking solution to each slide. Cover the slides gently with parafilm and incubate for 45 min at RT. Wash the slides three times in PBST for 5 min each.</li>
	<li>Drain excess PBST onto a paper towel. Add 50 &#181;L of antifade mounting medium onto each slide and cover with a coverslip. Image or store for no more than 48 h at -20 &#176;C.</li>
</ol>
4. Fiber Imaging by Fluorescence Microscopy
<ol>
	<li>Perform image acquisition through a 63X objective on an inverted microscope with an attached fully motorized filter wheel with FITC, Cy5 and Q Dot 655 detection. The excitation filter is 425 nm, and the emission filter is 655 nm with 20 nm centered at the emission peak 20.</li>
</ol>

<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 authors have nothing to disclose.

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><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, 100 mL</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&amp;nbsp;</td><td>Andor Technologies</td><td></td><td>with a Nitrogen pulsed laser&amp;nbsp;</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&amp;nbsp;</td><td>Sigma-Aldrich</td><td>17125</td><td></td></tr><tr><td>35 mm glass botomm plates 1.5 coverslip, 10 mm 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&amp;nbsp;</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 5,000</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>&amp;nbsp;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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

6.6K Views.  National Institute on Aging, National Institutes of Health. 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.

Video: Single Molecule Analysis of Laser Localized Psoralen Adducts

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.

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 ...

Cancer Research

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 ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biochemistry

Selective Area Modification of Silicon Surface Wettability by Pulsed UV Laser Irradiation in Liquid Environment

The wettability of silicon (Si) is one of the important parameters in the technology of surface functionalization of this material and fabrication of biosensing devices. We report on a protocol of using KrF and ArF lasers irradiating Si (001) samples immersed in a liquid environment with low number of pulses and operating at moderately low pulse fluences to induce Si wettability modification. Wafers immersed for up to 4 hr in a 0.01% H2O2/H2O solution did not show measurable change in their initial contact angle (CA) ~75&#176;. However, the 500-pulse KrF and ArF lasers irradiation of such wafers in a microchamber filled with 0.01% H2O2/H2O solution at 250 and 65 mJ/cm2, respectively, has decreased the CA to near 15&#176;, indicating the formation of a superhydrophilic surface. The formation of OH-terminated Si (001), with no measurable change of the wafer&#8217;s surface morphology, has been confirmed by X-ray photoelectron spectroscopy and atomic force microscopy measurements. The selective area irradiated samples were then immersed in a biotin-conjugated fluorescein-stained nanospheres solution for 2 hr, resulting in a successful immobilization of the nanospheres in the non-irradiated area. This illustrates the potential of&#160;the method for selective area biofunctionalization and fabrication of advanced Si-based biosensing architectures. We also describe a similar protocol of irradiation of wafers immersed in methanol (CH3OH) using ArF laser operating at pulse fluence of 65 mJ/cm2 and in situ formation of a strongly hydrophobic surface of Si (001) with the CA of 103&#176;. The XPS results indicate ArF laser induced formation of Si&#8211;(OCH3)x compounds responsible for the observed hydrophobicity. However, no such compounds were found by XPS on the Si surface irradiated by KrF laser in methanol, demonstrating the inability of the KrF laser to photodissociate methanol and create -OCH3 radicals.

We report on a process of in situ alteration of HF treated Si (001) surface into a hydrophilic or hydrophobic state by irradiating samples in microfluidic chambers filled with&#160;H2O2/H2O solution (0.01%-0.5%) or methanol solutions using pulsed UV laser of a relative low pulse fluence.

The wettability of silicon (Si) is one of the important parameters in the technology of surface functionalization of this material and fabrication of ...

Engineering

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 ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (&#9642;OH) to irreversibly label a protein&#8217;s solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of &#9642;OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.

This protocol presents a method to use inline radical dosimetry and a plasma light source to perform flash oxidation protein footprinting. This method replaces the hazardous UV laser to simplify and improve the reproducibility of fast photochemical oxidation of protein studies.

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes ...

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

Immunology and Infection

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

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. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

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 ...

Single Molecule Analysis of Laser Localized Psoralen Adducts

Summary

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Chapters in this video

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

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

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Selective Area Modification of Silicon Surface Wettability by Pulsed UV Laser Irradiation in Liquid Environment

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

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

Laser Micro-Irradiation to Study DNA Recruitment During S Phase