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 arbejde blev støttet af Natural Sciences and Engineering Research Council of Canada [Discovery grant 5026 til A.M] og af en næste Generation af forskere stipendium fra Cancer Research Society [21531 til A.M]. Vi takke Jean-François Lucier for at levere fremragende kvalitetskontrol af Fiji Python scripts og rådgivning på statistisk analyse. Vi takker Dr. Stephanie A. Yazinski for hvis fiksering løsning opskrift. Vi takke Paul-Ludovic Karsenti for hjælp med laser power målinger.

1. før sensibilisering af celler
<ol><li>24 timer før eksperimentet mikro-bestråling frø 40.000 U2OS celler pr. brønd i 1 x DMEM indeholdende 10% FBS i 8-godt kultur dias med 170 µm-tykke coverslip-lignende glas/polymer bunde til at sikre maksimal lystransmission 405 nm laser lys og fremragende billedbehandling inverteret Konfokal mikroskop med en laser-scanning. Hvis brug af en 8-godt microslide, skal du bruge 500 µL af medier eller løsninger pr. brønd for alle efterfølgende behandlinger og vaske trin i prot...

<ol>
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Ved hjælp af de metoder, der er skitseret ovenfor, tillader 405 nm laser mikro-bestråling af celler pre sensibiliseret med BBET eller BrdU den lokaliserede generation af DNA læsioner herunder DSBs inden for kerner af vedhængende humane celler. Kinetik af DDR på disse DSBs er lig dem genereret med andre metoder i eukaryote celler9,18,19. DSB resektion på mikro-bestrålet websteder fører til ssDNA-generation, som kan følge...

Celler er konstant udsat for endogene og eksogene kilder af DNA-skader, der truer deres genomisk integritet. DDR er et ensemble af signalering veje, der registrerer, signal og reparere DNA læsioner for at opretholde genom stabilitet. På DNA dobbelt-strenget pauser (DSBs), DDR sker hovedsageligt på to komplementære platforme: γ-H2A.X-mærket kromatin og en resektion enkeltstrenget DNA (ssDNA) region typisk belagt med ssDNA-bindende kompleks RPA1,2.
UV-laser mikro-bestråling af celler pre sensibiliseret med thymidine analoge 5-bromo-2' deoxyuridine (BrdU) eller bisbenzimide ethoxide trihydrochloride (BBET, Hoechst 33342) DNA farve skaber en blanding af DNA læsioner herunder enkelt-strenget pauser ( SSBs) og DSBs, som fremkalder en lokaliseret DDR på både kromatin og ssDNA platforme3,4. Tidligere arbejde viste, at rekruttering af genom vedligeholdelse faktorer til disse to forskellige platforme på DSB kan blive udsat for ved hjælp af høj energi UV-A lasere (335-365 nm) kombineret med IF2,5. Mikroskoper udstyret med disse lasere er dyre, da de kræver en høj energi laser og dedikeret UV-A fremsendende målene gør dem langt mindre udbredt i akademiske og farmaceutiske indstillinger end laser-scanning Konfokal mikroskoper med 405 nm laser linjer. Studiet af protein rekruttering og udvekslingen på mikro-bestrålet websteder er også udelukket af den besværlige manuelle billedanalyse forpligtet til at beskrive den dynamiske opførsel af genom vedligeholdelse faktorer.
Vi viser her, at før sensibilisering af celler med BrdU eller BBET nukleinsyre pletten efterfulgt af mikro-bestråling ved hjælp af en 405 nm laser på et fælles Konfokal mikroskop giver mulighed for overvågning af genom vedligeholdelse faktor dynamics på DNA læsioner. Ved hjælp af γ-H2A.X eller RPA komplekse underenheder som platform markører sammen med z-stacking til større dybdeskarphed og deconvolution for forbedret opløsning tillader eksperimentatoren at diskriminere faktorer, der rekrutteres lokalt til DSBs fra dem, der breder sig til store kromatin domæner omkring den oprindelige læsion. Denne sub klassificering i henhold til særskilte intra nukleare rum hjælper med at forfine de potentielle roller af uncharacterized proteiner rekrutteret til mikro-bestrålet websteder. Desuden, vi leverer praktisk protokoller og optimeret rørledninger for hurtigt at analysere dynamikken i genom vedligeholdelse faktorer ved hjælp af open source software Fiji (en ImageJ distribution)6,7,8. Disse justeringer til nuværende mikro-bestråling metoder flyveregler studiet af DDR i stort set ethvert laboratorium indstilling.

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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 skader svar (DDR) bruger en overflod af proteiner til at registrere, signal og reparere DNA læsioner. Afgrænse denne reaktion er afgørende for at forstå genom vedligeholdelse mekanismer. Da rekruttering og udvekslingen af proteiner på læsioner er meget dynamisk, kræver deres undersøgelse evnen til at generere DNA-skader på en hurtig og rumligt afgrænset måde. Her, beskrive vi procedurer for lokalt inducerer DNA-skader i humane celler ved hjælp af et almindeligt tilgængelige laser-scanning Konfokal mikroskop udstyret med en 405 nm laser linje. Ophobning af genom vedligeholdelse faktorer på laser striber kan vurderes ved immunofluorescens (IF) eller i realtid ved hjælp af proteiner markeret med fluorescerende journalister. Ved hjælp af fosforyleres Histon H2A. X (γ-H2A.X) og replikering Protein A (RPA) som markører, metoden giver nok opløsning til at diskriminere lokalt ansat faktorer fra dem, der spredes på tilstødende kromatin. Vi leverer yderligere ImageJ-baserede scripts til effektivt overvåge kinetik af protein relocalization i DNA skade websteder. Disse justeringer i høj grad forenkle studiet af DDR-dynamics.

Efter mikro-bestråling, celler er tilladt at inddrive for en bestemt periode afhængigt af arten af genom vedligeholdelse proteiner1,12. DSBs vil blive behandlet af nukleaser, mest udførligt under S og G2 faser i cellecyklus, til at oprette et begrænset område af ssDNA, som er hurtigt belagt af RPA og andre genom vedligeholdelse faktorer9. Denne ssDNA region er omgivet af kromatin, som er grundigt ændr...

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

At studere DNA skader reparation kinetik kræver et system til at fremkalde læsioner på definerede sub nukleare regioner. Vi beskriver en metode til at oprette lokaliserede dobbelt-strenget pauser ved hjælp af en laser-scanning Konfokal mikroskop udstyret med en 405 nm laser og give automatiserede procedurer for at kvantificere dynamikken i reparation faktorer på disse læsioner.

Undersøgelse af Protein rekruttering til DNA læsioner ved hjælp af 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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RNA-interferens-baserede Undersøgelse af funktion Heat Shock Protein 27 under hornhindeepitelkulturer Sårheling

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

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Laser Microirradiation at studere In Vivo cellulære svar til Simple og komplekse DNA-skader

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

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

Undersøgelse af RNA syntese ved hjælp af 5-Bromouridine mærkning og Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

En enkel, hurtig og kvantitative analysen til foranstaltning reparation af DNA-protein krydsbindinger på plasmider transfekteret i pattedyrceller

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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Sjælden begivenhed registrering ved hjælp af fejl-korrigeret DNA og RNA sekventering

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

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Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

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Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...