Name: investigation of protein recruitment to dna lesions using 405 nm laser micro-irradiation
Uploaded: 2018-03-20T15:00:00
Description: Watch this Scientific Journal Video about Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation at JoVE.com

Dette arbeidet ble støttet av naturvitenskap og Engineering Forskningsrådet Canada [Discovery grant 5026 til am] og en neste generasjon av forskere stipend fra Cancer Research Society [21531 til am]. Vi takker Jean-François Lucier for å gi utmerket kvalitetskontroll av Fiji Python-skript og råd om statistisk analyse. Vi takker Dr. Stephanie A. Yazinski for hvis fiksering løsning oppskriften. Vi takker Paul-Ludovic Karsenti for hjelp med laser makt mål.

1. pre sensibilisering celler
<ol><li>24 timer før mikro-bestråling eksperimentet, frø 40.000 U2OS celler per brønn i 1 x DMEM som inneholder 10% FBS i 8-brønnen lysbilder med 170 µm tykke dekkglassvæske som glass/polymer bunner å sikre maksimal transmisjon 405 nm laserlys og utmerket bildebehandling invertert AC confocal mikroskop med en laser-skanning. Hvis bruker en 8-og microslide, kan du bruke 500 µL av media eller løsninger per godt for alle etterfølgende behandlinger og vask trinnene av protokollen.<b...

<ol>
	<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> Molecular cell. 40 (2), 179-204 (2010).</li><li>Bekker-Jensen, 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=Spatial+organization+of+the+mammalian+genome+surveillance+machinery+in+response+to+DNA+strand+breaks.">Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks.</a> The Journal of Cell Biology. 173 (2), 195-206 (2006).</li><li>Limoli, A. C. L., Ward, J. F., May, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Method+for+Introducing+Double-Strand+Breaks+into+Cellular+DNA.">A New Method for Introducing Double-Strand Breaks into Cellular DNA.</a> Radiation Research. 134 (2), 160-169 (1993).</li><li>Polo, S. E., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+DNA+damage+response+proteins+at+DNA+breaks:+a+focus+on+protein+modifications.">Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications.</a> Genes &amp; development. 25 (5), 409-433 (2011).</li><li>Lukas, C., Falck, J., Bartkova, J., Bartek, J., Lukas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+spatiotemporal+dynamics+of+mammalian+checkpoint+regulators+induced+by+DNA+damage.">Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage.</a> Nature cell biology. 5 (3), 255-260 (2003).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+microscopy.">ImageJ for microscopy.</a> BioTechniques. 43 (1 Suppl), 25-30 (2007).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature methods. 9 (7), 676-682 (2012).</li><li>Symington, L. S., Gautier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+break+end+resection+and+repair+pathway+choice.">Double-strand break end resection and repair pathway choice.</a> Annual review of genetics. 45, 247-271 (2011).</li><li>Microirradiation Analysis Fiji Plugin. Available from: <a target="_blank" href="https://bitbucket.org/daniel_garneau/microirradiation_analysis/">https://bitbucket.org/daniel_garneau/microirradiation_analysis/</a> (2017)</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Izhar, 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=A+Systematic+Analysis+of+Factors+Localized+to+Damaged+Chromatin+Reveals+PARP-Dependent+Recruitment+of+Transcription+Factors.">A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.</a> Cell Reports. 11 (9), 1-15 (2015).</li><li>Panier, S., Boulton, 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=Double-strand+break+repair:+53BP1+comes+into+focus.">Double-strand break repair: 53BP1 comes into focus.</a> Nature Reviews Molecular Cell Biology. 15 (1), 7-18 (2013).</li><li>Mar&#233;chal, 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=PRP19+Transforms+into+a+Sensor+of+RPA-ssDNA+after+DNA+Damage+and+Drives+ATR+Activation+via+a+Ubiquitin-Mediated+Circuitry.">PRP19 Transforms into a Sensor of RPA-ssDNA after DNA Damage and Drives ATR Activation via a Ubiquitin-Mediated Circuitry.</a> Molecular Cell. 53 (2), 235-246 (2014).</li><li>Dubois, J. -. 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=A+phosphorylation-and-ubiquitylation+circuitry+driving+ATR+activation+and+homologous+recombination.">A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination.</a> Nucleic Acids Research. 45 (15), 8859-8872 (2017).</li><li>Wan, L., Huang, 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+PSO4+Complex+Associates+with+RPA+and+Modulates+the+Activation+of+ATR.">The PSO4 Complex Associates with RPA and Modulates the Activation of ATR.</a> The Journal of Biological Chemistry. 289 (10), 6619-6626 (2014).</li><li>Abbas, M., Shanmugam, I., Bsaili, M., Hromas, R., Shaheen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+human+Psoralen+4+(hPso4)+complex+in+replication+stress+and+homologous+recombination.">The role of the human Psoralen 4 (hPso4) complex in replication stress and homologous recombination.</a> The Journal of Biological Chemistry. 289 (20), 14009-14019 (2014).</li><li>Chung, W. H., Zhu, Z., Papusha, A., Malkova, A., Ira, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+resection+at+DNA+double-strand+breaks+leads+to+de+novo+telomere+formation+and+enhances+gene+targeting.">Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting.</a> PLoS Genetics. 6 (5), (2010).</li><li>Hicks, W. W. M., Yamaguchi, M., Haber, 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=Real-time+analysis+of+double-strand+DNA+break+repair+by+homologous+recombination.">Real-time analysis of double-strand DNA break repair by homologous recombination.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (8), 3108-3115 (2011).</li><li>Lackner, D. H., 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=A+generic+strategy+for+CRISPR-Cas9-mediated+gene+tagging.">A generic strategy for CRISPR-Cas9-mediated gene tagging.</a> Nature Communications. 6, 10237 (2015).</li><li>Ratz, M., Testa, I., Hell, S. W., Jakobs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+endogenous+protein+tagging+for+RESOLFT+super-resolution+microscopy+of+living+human+cells.">CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.</a> Scientific Reports. 5 (9592), (2015).</li><li>Linkert, M., 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=Metadata+matters:+Access+to+image+data+in+the+real+world.">Metadata matters: Access to image data in the real world.</a> Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Dinant, 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=Activation+of+multiple+DNA+repair+pathways+by+sub-nuclear+damage+induction+methods.">Activation of multiple DNA repair pathways by sub-nuclear damage induction methods.</a> Journal of Cell Science. 120 (15), 2731-2740 (2007).</li><li>Kong, X., 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=Comparative+analysis+of+different+laser+systems+to+study+cellular+responses+to+DNA+damage+in+mammalian+cells.">Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells.</a> Nucleic Acids Research. 37 (9), e68 (2009).</li><li>Holton, N. W., Andrews, J. F., Gassman, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Laser+Micro-irradiation+for+Examination+of+Single+and+Double+Strand+Break+Repair+in+Mammalian+Cells.">Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells.</a> Journal of Visualized Experiments. (127), (2017).</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> Bioconjugate Chemistry. 18 (2), 431-437 (2007).</li><li>Mortusewicz, O., Am&#233;, J. C., Schreiber, V., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback-regulated+poly(ADP-ribosyl)ation+by+PARP-1+is+required+for+rapid+response+to+DNA+damage+in+living+cells.">Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells.</a> Nucleic Acids Research. 35 (22), 7665-7675 (2007).</li><li>Kleiner, R. E., Verma, P., Molloy, K. R., Chait, B. T., Kapoor, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+proteomics+reveals+a+&#947;H2AX-53BP1+interaction+in+the+DNA+damage+response.">Chemical proteomics reveals a &#947;H2AX-53BP1 interaction in the DNA damage response.</a> Nature Chemical Biology. 11 (10), 807-814 (2015).</li><li>Soultanakis, R. P., 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=Fluorescence+detection+of+8-oxoguanine+in+nuclear+and+mitochondrial+DNA+of+cultured+cells+using+a+recombinant+Fab+and+confocal+scanning+laser+microscopy.">Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy.</a> Free Radical Biology and Medicine. 28 (6), 987-998 (2000).</li></ol>

Ved hjelp av metodene beskrevet ovenfor, kan 405 nm laser mikro-bestråling av celler pre sensibilisert med BBET eller BrdU lokalisert generering av DNA lesjoner inkludert DSBs innen kjerner i tilhenger menneskelige celler. Kinetics DDR på disse DSBs er lik de generert med andre metoder i eukaryote celler9,18,19. DSB reseksjon på mikro-bestrålt områder fører til ssDNA generasjon som kan følges med et RPA32 målretting anti...

Celler blir stadig utsatt for endogene og eksogene kilder til DNA skade som truer deres genomisk integritet. DDR er et ensemble av signalnettverk trasé som oppdage signal og reparere DNA lesjoner å opprettholde genomet stabilitet. DNA double-strandet pauser (DSBs), DDR oppstår hovedsakelig på to utfyllende plattformer: γ-H2A.X-merket chromatin og en resected single-strandet DNA (ssDNA) region vanligvis belagt med ssDNA-binding komplekse RPA1,2.
UV-laser mikro-bestråling av celler pre sensibilisert med thymidine analoge 5-bromo-2' deoxyuridine (BrdU) eller bisbenzimide ethoxide trihydrochloride (BBET, Hoechst 33342) DNA fargestoff skaper en blanding av DNA lesjoner inkludert single-strandet pauser ( SSBs) og DSBs som lokke fram en lokalisert DDR på både chromatin og ssDNA plattformer3,4. Tidligere arbeid viste at rekruttering genomet vedlikehold faktorer til disse to forskjellige plattformer på DSB kan bli diskriminert med høy energi UV-A lasere (335-365 nm) kombinert med Hvis2,5. Mikroskop utstyrt med slike lasere er kostbare som de krever en høy energi laser og dedikert UV-A overfører mål gjør dem langt mindre utbredt i akademiske og farmasøytiske enn laser-skanning AC confocal mikroskop med 405 nm laser linjer. Studiet av protein rekruttering og utveksling på mikro-bestrålt nettsteder er også utelukket av arbeidskrevende manuell bildeanalyse må beskrive den dynamiske oppførselen av genomet vedlikehold faktorer.
Her viser vi at den pre sensibilisering av celler med BrdU eller BBET nukleinsyre flekken etterfulgt av mikro-bestråling bruke en 405 nm laser på vanlige AC confocal mikroskop lar overvåking av genomet vedlikehold faktor dynamics på DNA lesjoner. Bruke γ-H2A.X eller RPA sammensatte underenheter som plattform markører sammen med z-stabling for større dybdeskarphet og deconvolution for forbedret oppløsning lar eksperimentator å diskriminere faktorer som rekrutteres lokalt til DSBs fra de som spres til store chromatin domener rundt den første leksjonen. Denne sub klassifisering i henhold til forskjellige intra kjernefysiske rom bidrar til å begrense potensielle rollene uncharacterized proteiner rekruttert til mikro-bestrålt nettsteder. Dessuten, vi tilbyr praktisk protokoller og optimalisert rørledninger for å raskt analysere dynamikken i genomet vedlikehold faktorer bruker åpen kilde-programvare Fiji (en ImageJ distribusjon)6,7,8. Disse avgrensninger til gjeldende mikro-bestråling metoder gjengi studiet av DDR i nesten enhver laboratorium setting.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Olympus FLUOVIEW FV3000 resonant laser scanning confocal microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:171px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">FluoView FV3000 software (version 2.1)</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">405 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td>Radiation source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Microscope incubator AIO-UNO-OLYUS-1</td>
			<td>Okolab</td>
			<td>OKO-UNO-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">60X /1.4 Objective</td>
			<td>Olympus</td>
			<td></td>
			<td>Oil immersion objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">8-well culture slides on coverglass (X-well)</td>
			<td>Sarstedt</td>
			<td>94.6190.802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">U2OS osteosarcoma human cell line</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td>Stable cell lines&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">4&#8217;,6-Diamidino-2-Phenylindole (DAPI)</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>Caution toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">5-bromo-2&#8242;-deoxyuridine&#160;(BrdU)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>59-14-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>30525-89-4</td>
			<td>Caution toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Bisbenzimide H 33342 (Hoechst 33342)</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Bovine serum albumin (BSA)</td>
			<td>ThermoFisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Trypsin EDTA 0.1%&#160;</td>
			<td>ThermoFisher</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Triton X-100&#160;</td>
			<td>BioShop</td>
			<td>TRX777.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Tween-20&#160;</td>
			<td>ThermoFisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Sucrose&#160;</td>
			<td>BioShop</td>
			<td>SUC507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">JetPRIME transfection reagent&#160;</td>
			<td>Polyplus</td>
			<td>114-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">ProLong Diamond Antifade mountant with DAPI&#160;</td>
			<td>ThermoFisher</td>
			<td>P36962</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">DMEM 1X, + phenol red</td>
			<td>Wisent&#160;</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">DMEM 1X, - phenol red</td>
			<td>Wisent</td>
			<td>319-051-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fetal bovine serum (FBS)</td>
			<td>Wisent&#160;</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Mouse anti-RPA32 antibody</td>
			<td>Abcam</td>
			<td>ab2175</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Rabbit anti-&#947;-H2A.X antibody</td>
			<td>Cell Signaling&#160;</td>
			<td>9718S</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Rabbit Anti-53BP1 antibody</td>
			<td>Cell Signaling&#160;</td>
			<td>4937S</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Rabbit Anti-PRP19 antibody</td>
			<td>Abcam</td>
			<td>ab27692</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Goat anti-rabbit IgG Alexa Fluor 647</td>
			<td>Cell Signaling&#160;</td>
			<td>4414S</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Goat anti-mouse&#160; IgG Alexa Fluor 488&#160;</td>
			<td>Cell Signaling&#160;</td>
			<td>4408S</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fiji image analysis open-source software&#160;</td>
			<td></td>
			<td></td>
			<td>https://fiji.sc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">1918-K Power meter&#160; with a 918D-SL-0D3 sensor</td>
			<td>Newport</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigation of protein recruitment to dna lesions using 405 nm laser micro-irradiation

DNA skade svar (DDR) bruker en mengde proteiner å oppdage signal og reparere DNA lesjoner. Skildre dette svaret er avgjørende å forstå genomet vedlikehold mekanismer. Siden rekruttering og utveksling av proteiner på lesjonene er svært dynamisk, krever studiet muligheten til å generere DNA skade på en rask og romlig tabulatordelte måte. Her beskriver vi fremgangsmåtene for å indusere lokalt DNA skade i menneskelige celler ved hjelp av et vanlig laserskanning AC confocal mikroskop utstyrt med en 405 nm laser linje. Akkumulering av genomet vedlikehold faktorene laser striper kan vurderes ved immunofluorescence (hvis) eller i sanntid med proteiner merket med fluorescerende journalister. Bruke fosforylert histone H2A. X (γ-H2A.X) og replikering Protein A (RPA) som markører, metoden gir tilstrekkelig oppløsning til å diskriminere lokalt rekruttert faktorer fra de som spredt på tilstøtende chromatin. Videre tilbyr vi ImageJ-baserte skript effektivt overvåke the kinetics av protein relocalization på DNA skade områder. Disse forbedringer forenkle studiet av dynamikken DDR.

Etter mikro-bestråling, celler kan gjenopprette for en bestemt tidsperiode avhengig av genomet vedlikehold proteiner1,12. DSBs vil bli behandlet av nucleases, mest omfattende i S og G2 faser av cellen syklusen, opprette et begrenset område av ssDNA som er raskt belagt av RPA og andre genomet vedlikehold faktorer9. Dette ssDNA området er omgitt av chromatin som er mye endret under DDR å regulere rekrutte...

Watch this Scientific Journal Video about Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation at JoVE.com

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

Studere DNA skade reparasjon kinetics krever et system å indusere lesjoner på definerte sub kjernefysiske regioner. Vi beskriver en metode for å opprette lokaliserte double-strandet bryter med laser-skanning AC confocal mikroskop utstyrt med en 405 nm laser og gi automatisert prosedyrer for å kvantifisere dynamikken i reparasjon faktorer på disse lesjoner.

Undersøkelse av Protein rekruttering DNA lesjoner med 405 Nm Laser mikro-bestråling

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

Research

JoVE Journal

Genetics

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The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

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