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

Arbetet i vårt labb stöds av KARRIÄR utmärkelse MCB0551798 från National Science Foundation och området bevilja R15GM86839 från NIGMS-NIH.

1. Tillväxt och UV-strålning. <ol><li> 200μl av en färsk natten kultur som innehåller plasmiden pBR322 odlas i Davis medelstora 1 kompletteras med 0,4% glukos, 0,2% casamino syror och 10 mikrogram / ​​ml tymin (DGCthy medium) och 100 mikrogram / ​​ml ampicillin är pelleterat. Cellpelleten sedan suspenderade i 200μl DGCthy medelstora saknas ampicillin och används för att inokulera 20 ml DGCthy medium. </li><li> Kulturer odlas utan ampicillin val i en skakande inkubator vid 37 ° C till en OD...

<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>

Typiska resultat från vildtyps celler i närvaro och frånvaro av UV-inducerade skador visas i figur 1. I avsaknad av skada, kan ~ 1% av den totala plasmid-DNA finns i Y-båge när celler växer snabbt i exponentiell fas. Efter bestrålning, är en övergående ökning av Y-formade molekyler observeras som blockerade replikering gafflar samlas på skadade områden. X-formade replikering intermediärer också övergående ackumuleras och kvarstår tills en tid som korrelerar med när skador repareras. <p class="jove...

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<td>An example of the Southern analysis of the 2D gel probed</td>
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<td>G15T8</td>
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<td>15 &mu;watt germicidal lamp</td>
<td>&nbsp;</td>
<td>Sylvania</td>
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<td>Blak-Ray UV Intensity Meter 254nm</td>
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<td>0.025 &mu;m pore disks</td>
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<td>Nylon membrane</td>
<td>&nbsp;</td>
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visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis

Felaktiga replikering i närvaro av DNA-skador är ansvarig för huvuddelen av cellulära omorganiseringar och mutagenes observerats i alla celltyper och är allmänt anses vara direkt förknippade med utveckling av cancer hos människor. DNA-skador, till exempel sådan som orsakas av UV-strålning, försämrar kraftigt möjligheterna för replikering för att duplicera genomiska mallen korrekt. Ett antal genprodukter har identifierats som är nödvändiga vid replikering möten DNA-skador i mallen. Har dock en kvarstående utmaningen varit att avgöra hur dessa proteiner processen lesioner vid replikering<em&gt; In vivo</em&gt;. Använda Escherichia coli som modellsystem, beskriver vi ett förfarande där två-dimensionella agarose-gel analys kan användas för att identifiera de strukturella intermediärer som uppstår på replikerar plasmider<em&gt; In vivo</em&gt; Efter UV-inducerade DNA-skador. Detta förfarande har använts för att påvisa att replikering gaffel blockerad av UV-inducerade skador genomgå en övergående omsvängning som stabiliseras av RecA och flera genprodukter i samband med RecF väg. Tekniken visar att dessa replikering intermediärer bibehålls tills en tid som korrelerar med avlägsnande av lesioner genom nukleotid excision reparation och återupptar replikering.

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 presenterar ett förfarande genom vilket tvådimensionella agarose-gel analys kan användas för att identifiera struktur replikering intermediärer som sker efter UV-bestrålning.

Visualisering av UV-inducerade replikering mellanprodukter i E. coli Med hjälp av två-dimensionell agaros-gel Analys

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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