Name: inducing a site specific replication blockage in e. coli using a fluorescent repressor operator system
Uploaded: 2016-08-21T15:00:00
Description: Watch this Scientific Journal Video about Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System at JoVE.com

This work was supported by the Australian Research Council [DP11010246].

1. Blocking Replication with FROS
<ol>
	<li>Inducing the Replication Blockage
	<ol>
		<li>Dilute a fresh overnight culture of an E. coli strain carrying a tetO array and pKM118 to OD600nm = 0.01 in a dilute complex medium (0.1% tryptone, 0.05% yeast extract, 0.1% NaCl, 0.17 M KH2PO4, 0.72 M K2HPO4) with antibiotics as required for selection. 
		Note: Do not add tetracycline for selection as it is not compatible with this system. Ma...

<ol>
	<li>Mirkin, E. V., Mirkin, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+stalling+at+natural+impediments.">Replication fork stalling at natural impediments.</a> Microbiol Mol Biol Rev. 71 (1), 13-35 (2007).</li><li>Gupta, M. K., 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=Protein-DNA+complexes+are+the+primary+sources+of+replication+fork+pausing+in+Escherichia+coli.">Protein-DNA complexes are the primary sources of replication fork pausing in Escherichia coli.</a> Proc Natl Acad Sci U S A. 110 (18), 7252-7257 (2013).</li><li>McGlynn, P., Guy, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+forks+blocked+by+protein-DNA+complexes+have+limited+stability+in+vitro.">Replication forks blocked by protein-DNA complexes have limited stability in vitro.</a> J Mol Biol. 381 (2), 249-255 (2008).</li><li>Tanner, N. 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=Single-molecule+studies+of+fork+dynamics+in+Escherichia+coli+DNA+replication.">Single-molecule studies of fork dynamics in Escherichia coli DNA replication.</a> Nat Struct Mol Biol. 15 (2), 170-176 (2008).</li><li>Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C., van Oijen, A. M. <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+replication+loops+reveal+temporal+control+of+lagging-strand+synthesis.">Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.</a> Nature. 457 (7227), 336-339 (2009).</li><li>Rudolph, C. J., Upton, A. L., Lloyd, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+stalling+and+cell+cycle+arrest+in+UV-irradiated+Escherichia+coli.">Replication fork stalling and cell cycle arrest in UV-irradiated Escherichia coli.</a> Genes Dev. 21 (6), 668-681 (2007).</li><li>Jeiranian, H. 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=Inefficient+replication+reduces+RecA-mediated+repair+of+UV-damaged+plasmids+introduced+into+competent+Escherichia+coli.">Inefficient replication reduces RecA-mediated repair of UV-damaged plasmids introduced into competent Escherichia coli.</a> Plasmid. 68 (2), 113-124 (2012).</li><li>Le Masson, M., Baharoglu, Z., Michel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ruvA+and+ruvB+mutants+specifically+impaired+for+replication+fork+reversal.">ruvA and ruvB mutants specifically impaired for replication fork reversal.</a> Mol Microbiol. 70 (2), 537-548 (2008).</li><li>Wang, X., Liu, X., Possoz, C., Sherratt, D. 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+two+Escherichia+coli+chromosome+arms+locate+to+separate+cell+halves.">The two Escherichia coli chromosome arms locate to separate cell halves.</a> Genes Dev. 20 (13), 1727-1731 (2006).</li><li>Lau, I. F., 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+and+temporal+organization+of+replicating+Escherichia+coli+chromosomes.">Spatial and temporal organization of replicating Escherichia coli chromosomes.</a> Mol Microbiol. 49 (3), 731-743 (2003).</li><li>Gordon, G. 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=Chromosome+and+low+copy+plasmid+segregation+in+E.+coli:+visual+evidence+for+distinct+mechanisms.">Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms.</a> Cell. 90 (6), 1113-1121 (1997).</li><li>Possoz, C., Filipe, S. R., Grainge, I., Sherratt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+of+controlled+Escherichia+coli+replication+fork+stalling+and+restart+at+repressor-bound+DNA+in+vivo.">Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo.</a> EMBO J. 25 (11), 2596-2604 (2006).</li><li>Payne, B. T., 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=Replication+fork+blockage+by+transcription+factor-DNA+complexes+in+Escherichia+coli.">Replication fork blockage by transcription factor-DNA complexes in Escherichia coli.</a> Nucleic Acids Res. 34 (18), 5194-5202 (2006).</li><li>Brewer, B. J., Fangman, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+localization+of+replication+origins+on+ARS+plasmids+in+S.+cerevisiae.">The localization of replication origins on ARS plasmids in S. cerevisiae.</a> Cell. 51 (3), 463-471 (1987).</li><li>Jeiranian, H. A., Schalow, B. J., 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=Visualization+of+UV-induced+replication+intermediates+in+E.+coli+using+two-dimensional+agarose-gel+analysis.">Visualization of UV-induced replication intermediates in E. coli using two-dimensional agarose-gel analysis.</a> J Vis Exp. (46), (2010).</li><li>Southern, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+specific+sequences+among+DNA+fragments+separated+by+gel+electrophoresis.">Detection of specific sequences among DNA fragments separated by gel electrophoresis.</a> J Mol Biol. 98 (3), 503-517 (1975).</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 (5609), 1064-1067 (2003).</li><li>Mettrick, K. A., Grainge, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+blocked+replication+forks+in+vivo.">Stability of blocked replication forks in vivo.</a> Nucleic Acids Res. , (2015).</li><li>Church, G. M., Gilbert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+sequencing.">Genomic sequencing.</a> Proc Natl Acad Sci U S A. 81 (7), 1991-1995 (1984).</li><li>Michel, B., Grompone, G., Flores, M. J., Bidnenko, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+process+stalled+replication+forks.">Multiple pathways process stalled replication forks.</a> Proc Natl Acad Sci U S A. 101 (35), 12783-12788 (2004).</li><li>Michel, B., Boubakri, H., Baharoglu, Z., LeMasson, M., Lestini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination+proteins+and+rescue+of+arrested+replication+forks.">Recombination proteins and rescue of arrested replication forks.</a> DNA Repair (Amst). 6 (7), 967-980 (2007).</li><li>Carl, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+mutants+with+temperature-sensitive+synthesis+of+DNA.">Escherichia coli mutants with temperature-sensitive synthesis of DNA.</a> Mol Gen Genet. 109 (2), 107-122 (1970).</li><li>Nawotka, K. A., Huberman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+gel+electrophoretic+method+for+mapping+DNA+replicons.">Two-dimensional gel electrophoretic method for mapping DNA replicons.</a> Mol Cell Biol. 8 (4), 1408-1413 (1988).</li><li>Cole, R. S., Levitan, D., Sinden, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+psoralen+interstrand+cross-links+from+DNA+of+Escherichia+coli:+mechanism+and+genetic+control.">Removal of psoralen interstrand cross-links from DNA of Escherichia coli: mechanism and genetic control.</a> J Mol Biol. 103 (1), 39-59 (1976).</li></ol>

During chromosome duplication, the replication machinery will encounter various impediments that prevent its progress. To ensure the entire single-origin chromosome is replicated, bacteria have numerous pathways for repair of the DNA that then enables the replisome to be reloaded20,21. Lesions, single stranded breaks, double stranded breaks and proteins tightly bound to the DNA may each be dealt with using a dedicated pathway, although there is likely to be significant overlap in these pathways. The most commo...

During DNA replication, the replisome faces obstacles on the DNA that impair its progression. DNA damage including lesions and gaps as well as aberrant structures can prevent the replisome from proceeding1. Recently, it has been found that proteins bound to the DNA are the most common source of impediment to replication fork progression2. The knowledge of the events following the encounter of the replisome with a nucleoprotein block has previously been limited by the inability to induce such a block in the chromosome of a living cell at a known location. In vitro analysis has enhanced our understanding of the kinetic behavior of an active replisome when it meets a nucleoprotein blockage3, as well as the mechanistic details of the replisome itself4,5. Current understanding of the repair of replication is generally undertaken with UV as the damaging agent and studied using plasmid DNA in vivo6-8. While the proteins that may be involved in repair of DNA after it encounters an in vivo nucleoprotein block are generally understood from these studies, whether there are variations in the molecular events within the repair pathways owing to the distinct cause in the replication block is still yet to be determined.
Here, we describe a system that allows a nucleoprotein block to be established in a specific location of the chromosome using a Fluorescent Repressor Operator System (FROS). We utilize a strain of E. coli that has had an array of 240 tetO sites incorporated into the chromosome9. Each tetO site within the array has a 10 bp random sequence flanking it to increase the stability of the array by preventing RecA-mediated recombination within the array. This array, and variations of it, were originally used to understand E. coli chromosome dynamics10,11 but were then adapted to prevent in vivo replication12. The array has been found to be stably maintained and to block close to 100% of replication forks when bound by TetR10,12. The use of the similar lacO array in vitro has found as few as 22 sites were sufficient to block 90% of replication, although this shorter array was less effective in vivo13. To adapt the array to create a nucleoprotein blockage, the repressor protein must be highly overproduced under optimized conditions where it then binds to the array to create a roadblock. The formation of the blockage, and its subsequent release, can be monitored through the use of fluorescence microscopy if a fluorescently tagged variant of the Tet repressor is used. The status of replication in each cell is indicated by the number of foci seen, where a single focal point means only one copy of the array is present within the cell and multiple foci are indicative of active replication. This active replication is enabled when the nucleoprotein blockage is reversed by the addition of the gratuitous inducer that decreases the binding affinity of TetR for the operator site sufficiently for the replisome to proceed through the array. The repressor protein is still able to bind to the DNA with high enough affinity that the now multiple copies of the array can be visualized.
More intricate details of the events at the nucleoprotein blockage can be discovered using neutral-neutral two-dimensional agarose gel electrophoresis and Southern hybridization14-16. These techniques allow the analysis of DNA structures across the population. The replication intermediates that are formed during the event, and potentially remain unrepaired, can be visualized. By varying the restriction enzyme and probe utilized, the intermediates can be visualized not only in the array region but also upstream of the array when the replication fork regresses17,18. The regression takes place subsequent to the replisome dissociation; the leading and lagging nascent strands separate from the template strands and anneal to each other as the template strands concurrently re-anneal resulting in a four-way DNA structure (a Holliday junction).
Using this system it has been shown that the replication fork is not stable when it encounters this block18. In addition, temperature sensitive derivatives of replisome components can be utilized to prevent reloading of the replication fork once it has collapsed. Once a block is established, the strain can be shifted to a non-permissive temperature to ensure a synchronous deactivation of the replisome and a controlled prevention of reloading. This temperature-induced deactivation ensures all of the forks within the population have collapsed at a given time and allows the assessment of what happens when the replisome collapses, how the DNA is processed, and what is required to restart the process of DNA replication.
An advantage of the system described here is that the nucleoprotein block is fully reversible; therefore, the ability of the cells to recover from the nucleoprotein block is able to be followed. The addition of anhydrotetracycline to the cells will relieve the tight binding of the repressor, allowing a replication fork to proceed through and the cell to regain viability. The relief of the blockage can be visualized by neutral-neutral two-dimensional agarose gel electrophoresis after 5 min, and by microscopy within 10 min. Furthermore, viability analysis can reveal the ability of the strain to recover from the replication blockage and continue to proliferate.
By altering the genetic background of the strains used in the experimental procedure described here, the repair pathways for this type of blockage can be elucidated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tryptone</td>
			<td>Sigma-Aldrich</td>
			<td>16922</td>
			<td>Growth media component</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>VWR</td>
			<td>27810.364</td>
			<td>Growth media component</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma-Aldrich</td>
			<td>92144</td>
			<td>Growth media component</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td>Growth media component; Potassium buffer component</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td>Growth media component; Potassium buffer component</td>
		</tr>
		<tr>
			<td>L-Arabinose</td>
			<td>Sigma-Aldrich</td>
			<td>A3256</td>
			<td>For induction of TetR-YFP production</td>
		</tr>
		<tr>
			<td>Anhydrotetracycline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>37919</td>
			<td>Release of replication bloackage</td>
		</tr>
		<tr>
			<td>Axioskop 2 Fluorescence microscope</td>
			<td>Zeiss</td>
			<td>452310</td>
			<td>Visualization of cells</td>
		</tr>
		<tr>
			<td>eYFP filter set</td>
			<td>Chroma Technology</td>
			<td>41028</td>
			<td>Visualization of YFP</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Hamamatsu</td>
			<td>Orca-AG</td>
			<td>Visualization of cells</td>
		</tr>
		<tr>
			<td>MetaMorph&#160; Software (Molecular Devices)</td>
			<td>SDR Scientific</td>
			<td>31282</td>
			<td>Version 7.8.0.0 used in the preparation of this manuscript</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioline</td>
			<td>BIO-41025</td>
			<td>For agarose plugs and gel electrophoresis</td>
		</tr>
		<tr>
			<td>Original Glass Water Repellent (Rain-X)</td>
			<td>Autobarn</td>
			<td>DIO1470</td>
			<td>For agarose plug manufacture</td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>VWR</td>
			<td>VWRC103157P</td>
			<td>TE, TBE buffer component</td>
		</tr>
		<tr>
			<td>Ethylene diaminetetraacetic acid</td>
			<td>Ajax Finechem</td>
			<td>AJA180</td>
			<td>0.5 M EDTA disodium salt solution adjusted to pH 8.0 with NaOH.</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Bacteriostatic agent</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>258148</td>
			<td>TE buffer component</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td>Cell lysis buffer component</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt (Sarkosyl)</td>
			<td>Sigma-Aldrich</td>
			<td>L5125</td>
			<td>Cell lysis and ESP buffer component</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma-Aldrich</td>
			<td>R6513</td>
			<td>Cell lysis buffer component</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Amresco</td>
			<td>6300</td>
			<td>Cell lysis buffer component</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Amresco</td>
			<td>AM0706</td>
			<td>ESP buffer component</td>
		</tr>
		<tr>
			<td>Sub-Cell Model 192 Cell</td>
			<td>BioRad</td>
			<td>1704507</td>
			<td>Electrophoresis system</td>
		</tr>
		<tr>
			<td>UV transilluminator 2000</td>
			<td>BioRad</td>
			<td>1708110</td>
			<td>Visualization of DNA</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>BioRad</td>
			<td>1610433</td>
			<td>Visualization of DNA</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>VWR</td>
			<td>PROL20185.360</td>
			<td>TBE component</td>
		</tr>
		<tr>
			<td>Hybond-XL nylon memrbane</td>
			<td>Amersham</td>
			<td>RPN203S</td>
			<td>Zeta-Probe Memrbane (BioRad 1620159) can also be used</td>
		</tr>
		<tr>
			<td>3MM Whatman chromatography paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3030690</td>
			<td>Southern blotting</td>
		</tr>
		<tr>
			<td>HL-2000 Hybrilinker</td>
			<td>UVP</td>
			<td>95-0031-01/02</td>
			<td>Crosslinking of DNA and hybridization</td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acid from salmon sperm</td>
			<td>Sigma-Aldrich</td>
			<td>31149</td>
			<td>Hybridization buffer component</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td>Denaturation buffer component</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>VWR</td>
			<td>PROL27833.363</td>
			<td>Transfer buffer</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate (SDS)</td>
			<td>Amresco</td>
			<td>227</td>
			<td>Wash buffer component</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td>Hybridization buffer component</td>
		</tr>
		<tr>
			<td>Random Hexamer Primers</td>
			<td>Bioline</td>
			<td>BIO-38028</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow fragment</td>
			<td>New England BioLabs</td>
			<td>M0212L</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Set</td>
			<td>Bioline</td>
			<td>BIO-39025</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate-32P-ATP</td>
			<td>PerkinElmer</td>
			<td>BLU502A</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage Phosphor Screen</td>
			<td>GE Healthcare Life Sciences</td>
			<td>GEHE28-9564-76</td>
			<td>BAS-IP MS 3543 E multipurpose standard 35x43cm screen</td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9558-09</td>
			<td>Visualization of blot</td>
		</tr>
		<tr>
			<td>Hybridization bottle</td>
			<td>UVP</td>
			<td>07-0194-02</td>
			<td>35 x 300mm</td>
		</tr>
	</tbody>
</table>

inducing a site specific replication blockage in e. coli using a fluorescent repressor operator system

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

The FROS is an inducible, site-specific nucleoprotein block that enables replication intermediates to be visualized in living cells12,18. A general experimental design for sampling cells is illustrated in Figure 1. The timing of the sampling and variations in genetic background make this a versatile system for studying the repair of such a block. The schematic illustrates how temperature sensitive mutants, such as dnaBts and dnaCts that have b...

Watch this Scientific Journal Video about Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System at JoVE.com

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

The overall goal of this experiment is to stall a replication fork at a nucleoprotein block and observe the DNA structures at the site of the block for the purpose of studying DNA repair pathways. This method can help answer key questions about DNA replication and repair, such as how the DNA is processed when the cell's replication apparatus encounters a protein roadblock. The main advantage of this technique is that we can reversibly stall a replication fork at a specific location on the chromosome across an entire population of living cells.Demonstrating this procedure will be the following members of my laboratory, Dr.Karla Mettrick, and graduate student, Georgia Weaver, and Tayla-Ann Corocher. An E.coli strain carrying a tetracycline operator array in the pKM1 plasmid is used for this procedure. pKM1 encodes for TetR-YFP, an inducible yellow fluorescent protein-tagged tetracycline repressor protein.Dilute a fresh overnight culture of this E.coli strain to an optical density at 600 nanometers of 0.01 in a dilute complex medium with antibiotics as required for selection. Grow the culture at 30 degrees celsius with shaking to an optical density at 600 nanometers of 0.05 to 0.1. Remove a 10 milliliter sample to serve as the uninduced control.Add 0.01%arabinose to the remaining culture to induce the production of TetR-YFP from pKM1. Continue to grow both the uninduced and induced cultures at 30 degrees celsius with shaking. After one hour, check for the presence of a single focal point within each cell of the induced culture using fluorescence microscopy.If the induced cells are confirmed blocked, indicated by more than 70%of cells having a single focus, record the optical density and remove 7.5 milliliters of the sample for analysis by two-dimensional gel electrophoresis. Remove an equivalent sample from the uninduced control culture. Prior to fluorescence microscopy, prepare the agarose pads for preventing movement of the cells during visualization.For each agarose pad, pipette 500 microliters of molten agarose onto a microscope slide and overlay the coverslip immediately before the agarose solidifies. Store the slides between wet tissues at four degrees celsius until needed. When it is time to check the cells, remove the coverslip from the agarose pad and pipette 10 microliters of bacterial culture onto the center of the pad.After approximately five minutes, when the agarose pad has dried, replace the coverslip. Place a drop of immersion oil onto the coverslip and place the slide under the optics. Visualize the cells using phase microscopy at 100x magnification.On the Acquire dialog box within the imaging software, set Exposure Time to 100 milliseconds. Select Acquire to capture the image. Do not move the stage.In the Acquired dialog box, select YFP as the illumination for the external shutter linked to the camera. Set the Exposure Time to 1, 000 milliseconds. Turn off the stage light and select Acquire to capture the fluorescence image.To begin this procedure, add sodium azide to previously-collected culture samples to a final concentration of 0.1%and incubate on ice for at least five minutes. Centrifuge the cells at 5, 000 times g for ten minutes. Discard the supernatant.Resuspend each cell pellet in 200 microliters of PIV buffer and transfer the cells to a microfuge tube. Microcentrifuge to pellet the cells and discard the supernatant. Treat a microscope slide with 40 microliters of an appropriate glass water repellent or silicone solution.Rub the slide with a tissue until it is dry. Resuspend the cells with the lowest cell density in 50 microliters of PIV and place at 50 degrees celsius in a heat block. For all other samples, adjust the volumes of PIV so the final cell density is the same in each tube.Add an equal volume of freshly-prepared 0.8%Agarose solution in PIV to the samples. Keep the samples in the heat block to ensure they are above 50 degrees celsius to prevent solidification. Place 20 microliter drops of the cell-agarose suspension on the treated slide to produce plugs that are hemispherical.When the plugs have solidified, gently slide all of the plugs from one sample into a single microfuge tube and add one milliliter of cell lysis buffer. Incubate at 37 degrees celsius for two hours. After two hours, remove the cell lysis buffer and add 1 milliliter of EDTA-Sarkosyl-Proteinase K or ESP solution.Incubate at 50 degrees celsius overnight or until the plugs are transparent. Once the plugs are transparent, remove the ESP buffer and transfer the plugs to a 15-milliliter tube. Add 12 milliliters of TE buffer and let stand for 30 minutes.Wash the plugs a total of five times with TE buffer. Store the plugs at four degrees celsius in one milliliter of TE buffer. To analyze the replication intermediates, the DNA in the agarose plugs is digested with an appropriate restriction enzyme for the region of interest.In this example, EcoRV cuts the DNA immediately before the array, within the array, and after the array, to give 5.5 kb and 6.7 kb fragments in the region of interest. To begin this procedure, transfer an agarose plug to a fresh microfuge tube and add 150 microliters of restriction enzyme buffer. Add 25 to 100 units of restriction enzyme and digest at 37 degrees celsius for six to eight hours.Prepare a 0.4%agarose gel at four degrees celsius. Pour 300 milliliters of molten agarose into a gel tray of approximately 25 by 25 centimeters. Insert a comb to make the wells approximately the same width as the diameter of the plug.When the gel is set, remove the comb and move the gel to room temperature. Using an inoculation loop, slide one of the digested agarose plugs into a well, positioning the flat side of the plug against the side of the well where the DNA will enter the gel. Insert a single plug in the same manner into every second well.Pipette molten 0.4%agarose into the wells to seal the plugs in position. Load a one kb DNA ladder in an empty well, leaving a gap between the ladder and samples. Run the gel overnight in 1xTBE at room temperature.On the following day, stain the gel in a water bath containing 0.3 micrograms per milliliter of ethidium bromide for 20 minutes. Visualize the DNA with a long-wave UV transilluminator and cut out each lane fragment with a straight even cut minimizing the inclusion of excess agarose on either side of the lane. Place the excised gel lane in a gel tray at 90 degrees to the direction of DNA migration.The next step is to prepare 300 milliliters of agarose for the second dimension gel. When the agarose is cooled to 50 degrees celsius, pipette the molten agarose around the gel slices to solidify their position. Pour the remaining agarose into the tray to a depth that is at least level with the gel slices.Once the gel is solidified, electrophorese it in 1xTBE containing 0.3 micrograms per milliliter of ethidium bromide at four degrees celsius until the DNA has migrated approximately 10 centimeters. Excise the blocks where the genomic DNA is present including the gel above it, to include the DNA that is not visible. The DNA within the gel is subsequently visualized by Southern hybridization as described in the text protocol.In this system, a replication block was confirmed by a majority of cells containing one focus corresponding to one copy of the array within the cell. The addition of anhydrotetracycline reversed the block and subsequent duplication of the array was visualized as the accumulation of cells with multiple foci. Cells with blocked replication also showed growth inhibition that was reversed by subsequent growth in the presence of anhydrotetracycline.To analyze the replication intermediates, the DNA was electrophoresed in the first dimension. The DNA of interest was then excised and a second dimension electrophoresis resulted in a diagonal of linear DNA. Southern hybridization of the array DNA revealed two spots in the unblocked sample corresponding to the expected 5.5kb and 6.7kb fragments of the array.The blocked sample showed a decrease in the 5.5kb spot and the addition of an elliptical signal indicating an accumulation of Y-shaped DNA. Addition of anhydrotetracycline eliminated the Y-shaped DNA signal. Another common intermediate is the Holliday junction visualized as a cone signal at the top of the Y-arc and a spike from the linear DNA at the end of the Y-arc.Once mastered, this technique can be done in eight days if it is preformed properly. While a 2D procedure, it is important to remember that the quality of the DNA plugs is critical for the optimal visualization of the DNA structures.

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