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

Lavoro nel nostro laboratorio è supportato da premio CARRIERA MCB0551798 dalla National Science Foundation e AREA concedere R15GM86839 dal NIGMS-NIH.

1. Crescita e di irradiazione UV. <ol><li> 200μl di una cultura fresco durante la notte contenente il plasmide pBR322 cresciuto in media 1 Davis integrato con 0,4% di glucosio, 0,2% di acidi casamino, e 10 mg / ml timina (media DGCthy) e 100 mg / ml ampicillina è pellet. Il pellet cellulare viene risospeso in medium DGCthy 200μl manca ampicillina e utilizzati per inoculare 20 ml di medium DGCthy. </li><li> Le culture sono cresciute senza selezione ampicillina in una agitazione incubatore a 37 ° C ad un ...

<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>

I risultati tipici ottenuti da cellule di tipo selvatico in presenza e in assenza di danno indotto dai raggi UV sono mostrati in figura 1. In assenza di danni, ~ 1% del DNA totale plasmide si possono trovare nell&#39;arco Y quando le cellule sono in rapida crescita in fase esponenziale. A seguito di irradiazione, un aumento transitorio delle molecole a forma di Y si osserva come le forcelle replica bloccato accumulano nei siti danneggiati. La forma di X intermedi replica anche transitoriamente si accumulano e persistono...

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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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<tr>
<td>15 &mu;watt germicidal lamp</td>
<td>&nbsp;</td>
<td>Sylvania</td>
<td>F20T12/GO</td>
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<tr>
<td>Blak-Ray UV Intensity Meter 254nm</td>
<td>&nbsp;</td>
<td>Daigger</td>
<td>EF28195T</td>
<td>UVC photometer</td>
</tr>
<tr>
<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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<tr>
<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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<tr>
<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

Replica inesatte in presenza di danno al DNA è responsabile della maggior parte dei riarrangiamenti cellulare e mutagenesi osservato in tutti i tipi di cellule ed è ampiamente creduto di essere direttamente associati con lo sviluppo del cancro negli esseri umani. Danno al DNA, come quella indotta da radiazioni UV, limita fortemente la capacità di replicazione di duplicare il modello di genomica con precisione. Un certo numero di prodotti genici sono stati identificati, che sono necessari quando la replicazione incontra le lesioni del DNA nel modello. Tuttavia, una sfida che rimane è stato quello di determinare come queste lesioni processo di proteine ​​durante la replica<em&gt; In vivo</em&gt;. Utilizzando Escherichia coli come sistema modello, si descrive una procedura in cui bidimensionale agarosio-gel analisi può essere utilizzato per identificare gli intermedi strutturali che sorgono su plasmidi replicare<em&gt; In vivo</em&gt; Segue indotte dai raggi UV danno al DNA. Questa procedura è stata utilizzata per dimostrare che le forcelle replica bloccato dal danno indotto dai raggi UV subiscono un&#39;inversione transitoria che è stabilizzata da Reca e dei prodotti genici diversi relativi alla via RECF. La tecnica dimostra che questi intermedi replica vengono mantenute fino a un tempo che si correla con la rimozione delle lesioni da riparazione per escissione dei nucleotidi e riprende la replica.

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

Vi presentiamo una procedura che bidimensionale agarosio-gel analisi può essere utilizzato per identificare la struttura degli intermedi di replica che si verificano dopo irradiazione UV.

Visualizzazione dei raggi UV-indotta Intermedi replica in E. coli Utilizzando bidimensionale agarosio-gel Analisi

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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