Name: visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis
Uploaded: 2010-12-21T16:00:00
Description: Watch this Scientific Journal Video about Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis at JoVE.com

Werken in ons lab wordt ondersteund door LOOPBAAN award MCB0551798 van de National Science Foundation en het gebied te verlenen R15GM86839 van de NIGMS-NIH.

1. Groei en UV-straling. <ol><li> 200μl van een verse &#39;s nachts de cultuur die het plasmide pBR322 gekweekt in Davis medium 1 aangevuld met 0,4% glucose, 0,2% casamino zuren, en 10 ug / ml thymine (DGCthy medium) en 100 ug / ml ampicilline is gekorreld. De cel pellet wordt vervolgens geresuspendeerd in 200μl DGCthy medium zonder ampicilline en wordt gebruikt om 20 ml DGCthy medium enten. </li><li> Culturen zijn geteeld zonder ampicilline selectie in een incubator schudden bij 37 ° C tot een OD 6...

<ol>
	<li>Davis, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Isolation+of+Biochemically+Deficient+Mutants+of+Bacteria+by+Means+of+Penicillin.">The Isolation of Biochemically Deficient Mutants of Bacteria by Means of Penicillin.</a> Proc Natl Acad Sci U S A. 35, 1-10 (1949).</li><li>Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. T. <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-Induced+Replication+Fork+Regression+and+Processing+in+Escherichia+coli.">DNA Damage-Induced Replication Fork Regression and Processing in Escherichia coli.</a> Science. 299, 1064-1067 (2003).</li><li>Friedman, K. L., Brewer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+replication+intermediates+by+two-dimensional+agarose+gel+electrophoresis.">Analysis of replication intermediates by two-dimensional agarose gel electrophoresis.</a> Methods Enzymol. 262, 613-627 (1995).</li><li>Spivak, G., Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+damage+and+repair+in+specific+DNA+sequences.">Determination of damage and repair in specific DNA sequences.</a> METHODS: A Companion to Methods in Enzymology. 7, 147-161 (1995).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning - A laboratory manual. , (2001).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvABC+is+required+to+resolve+holliday+junctions+that+accumulate+following+replication+on+damaged+templates+in+Escherichia+coli.">RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli.</a> J Biol Chem. 281, 28811-28821 (2006).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvAB+and+RecG+Are+Not+Essential+for+the+Recovery+of+DNA+Synthesis+Following+UV-Induced+DNA+Damage+in+Escherichia+coli.">RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli.</a> Genetics. 166, 1631-1640 (2004).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecO+Acts+with+RecF+and+RecR+to+Protect+and+Maintain+Replication+Forks+Blocked+by+UV-induced+DNA+Damage+in+Escherichia+coli.">RecO Acts with RecF and RecR to Protect and Maintain Replication Forks Blocked by UV-induced DNA Damage in Escherichia coli.</a> J Biol Chem. 279, 3492-3496 (2004).</li><li>Belle, J. J., Casey, A., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+DnaB+helicase+leads+to+the+collapse+and+degradation+of+the+replication+fork:+a+comparison+to+UV-induced+arrest.">Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.</a> J Bacteriol. 189, 5452-5462 (2007).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecBCD+and+RecJ/RecQ+initiate+DNA+degradation+on+distinct+substrates+in+UV-irradiated+Escherichia+coli.">RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli.</a> Radiat Res. 168, 499-506 (2007).</li><li>Al-Hadid, Q., Ona, K., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecA433+cells+are+defective+in+recF-mediated+processing+of+disrupted+replication+forks+but+retain+recBCD-mediated+functions.">RecA433 cells are defective in recF-mediated processing of disrupted replication forks but retain recBCD-mediated functions.</a> Mutat Res. 645, 19-26 (2008).</li></ol>

Typische resultaten van wild-type cellen in de aanwezigheid en afwezigheid van UV-geïnduceerde schade zijn weergegeven in figuur 1. Bij het ontbreken van schade, kan ~ 1% van de totale plasmide DNA te vinden in de Y-boog wanneer de cellen groeien snel in exponentiële fase. Na de bestraling, is een tijdelijke toename van Y-vormige moleculen waargenomen als geblokkeerd replicatie vorken zich ophopen aan beschadigd sites. De X-vormige replicatie tussenproducten ook tijdelijk ophopen en blijven bestaan ​​totdat er een...

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<td>An example of the Southern analysis of the 2D gel probed</td>
<td>&nbsp;</td>
<td>GE</td>
<td>G15T8</td>
<td>Yellow Lighting</td>
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<td>15 &mu;watt germicidal lamp</td>
<td>&nbsp;</td>
<td>Sylvania</td>
<td>F20T12/GO</td>
<td>UV Lamp</td>
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<td>Blak-Ray UV Intensity Meter 254nm</td>
<td>&nbsp;</td>
<td>Daigger</td>
<td>EF28195T</td>
<td>UVC photometer</td>
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<td>0.025 &mu;m pore disks</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>VSWP04700</td>
<td>Floating dialysis disks</td>
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<td>PvuII</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>ER0632</td>
<td>Restriction Endonuclease</td>
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<td>Nick-translation kit</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>976776</td>
<td>To make 32P-labeled probe</td>
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<td>Blotting Paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-704</td>
<td>For Southern transfer</td>
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<td>Nylon membrane</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>RPN203S</td>
<td>For Southern transfer</td>
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visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis

Onnauwkeurige replicatie in de aanwezigheid van DNA-schade is verantwoordelijk voor het merendeel van de cellulaire herschikkingen en mutagenese waargenomen in alle celtypes en wordt algemeen aangenomen dat direct geassocieerd met de ontwikkeling van kanker bij de mens. DNA-schade, zoals die veroorzaakt door UV-straling, vermindert sterk het vermogen van replicatie naar de genomische template nauwkeurig te dupliceren. Een aantal van gen-producten zijn geïdentificeerd die nodig zijn voor het replicatie ontmoetingen DNA laesies in de sjabloon. Er is echter een resterende uitdaging is hoe deze eiwitten te bepalen laesies tijdens replicatie<em&gt; In vivo</em&gt;. Met behulp van Escherichia coli als een modelsysteem, beschrijven we een procedure waarin twee-dimensionale agarose-gel-analyse kan worden gebruikt om de structurele tussenproducten die ontstaan ​​bij replicerende plasmiden te identificeren<em&gt; In vivo</em&gt; Na UV-geïnduceerde DNA-schade. Deze procedure is gebruikt om aan te tonen dat de replicatie vorken geblokkeerd door UV-geïnduceerde schade een tijdelijke omkering die wordt gestabiliseerd door RecA en verschillende genproducten worden geassocieerd met het RecF pad ondergaan. De techniek laat zien dat deze replicatie tussenproducten worden gehandhaafd tot een moment dat correleert met de verwijdering van de letsels door nucleotide excisie reparatie en replicatie hervat.

Watch this Scientific Journal Video about Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis at JoVE.com

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We presenteren een procedure waarbij twee-dimensionale agarose-gel analyse kan worden gebruikt om de structuur van de replicatie tussenproducten die optreden na UV-straling te identificeren.

Visualisatie van UV-geïnduceerde Replicatie Intermediates in E. coli Met behulp van twee-dimensionale Agarose gel-analyse

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

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Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

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