The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for funding this work.

1. Preparation of Media and Cultures
<ol>
	<li>For LB + Amp: Prepare 500 ml of Luria-Bertani broth (LB) sterilized using an autoclave at 121 &#176;C for 30 min. Supplement with ampicillin to yield a final concentration of 100 &#181;g/ml of ampicillin and store at 4 &#176;C until required.</li>
	<li>For TB + Amp: Prepare for large (500 ml or greater) volumes of ministring production. Prepare 600 ml of sterile Terrific Broth (TB) sterilized using an autoclave at 121 &#176;C for 30 min. Supplement with ampicillin to yield a final concentration of 100 &#181;g/ml of ampicillin and store at 4 &#176;C until required.</li>
	<li>For LB agar + Amp: Prepare sterile LB agar plates by preparing LB + Amp supplemented with 15 g/L of agar. Aliquot approximately 15 - 20 ml in sterile Petri dishes and store upside down at 4 &#176;C until required.</li>
	<li>Prepare a streak plate of W3NN[pDMS] by aseptically streaking W3NN[pDMS] cells on LB agar + Amp. Incubate the plate overnight at 30 &#176;C, until single colonies can be observed. Do not incubate W3NN above this temperature to avoid derepressing protelomerase expression.</li>
	<li>Aseptically transfer 5 ml of LB + Amp into a sterile test tube. Prepare an overnight culture of W3NN[pDMS] by inoculating this volume with a single colony from the streak plate. Incubate the culture overnight at 30 &#176;C in a shaker at 230-250 rpm.</li>
</ol>
2. Growth Phase
<ol>
	<li>Aseptically transfer 10 ml of LB + Amp into a sterile 125 ml Erlenmeyer flask and inoculate with 100 &#181;l of the overnight W3NN[pDMS] culture into the prepared LB + Amp, making a 1:100 dilution.
	<ol>
		<li>For larger volumes, add 500 &#181;l of overnight culture into 50 ml of TB + Amp in a 250 ml Erlenmeyer flask, instead of LB + Amp.</li>
		<li>Alternatively, use as the second &#34;representative&#34; culture in step 2.2.</li>
	</ol>
	</li>
	<li>Incubate in a shaking incubator at 30 &#176;C and 250 rpm until the culture reaches mid to late log phase, indicated by an optical density (OD) or absorbance: A600 = 0.8. After 1-2 hr, the medium should begin to appear turbid to the naked eye.
	<ol>
		<li>Aseptically transfer 1 ml of culture to a clean spectrophotometer cuvette.</li>
		<li>Measure the light absorbance using a UV-vis spectrophotometer with the wavelength set to 600 nm. Use 1 ml of the media in which cells were grown to set the blank (LB or TB). Optionally, use the representative culture from step 2.1.3 for this step instead.</li>
		<li>If the culture has not yet reached the appropriate OD, return to the shaker and check once again every half hour. Check the OD more frequently as the A600 approaches 0.8.</li>
	</ol>
	</li>
	<li>Once the culture has reached late log phase, indicated by A600 = 0.8, aseptically transfer the culture into sterile 50 ml centrifuge tubes.</li>
	<li>Collect cells by centrifuging the culture at 10,000 x g for 10 min. Inspect for a pellet at the bottom of the tube.</li>
	<li>Decant the cleared supernatant into an appropriate biohazard waste container. Do not disturb the cell pellet.</li>
	<li>Repeat steps 2.4-2.5 until all cells have been collected.</li>
	<li>Re-suspend the pellet in 100 &#181;l LB + Amp. Aspirate using a micropipette or use a vortex mixer. For greater ministring production, re-suspend the pellet from a 50 ml TB + Amp culture into 1 ml of fresh LB + Amp media.</li>
</ol>
3. Ministring Production
<ol>
	<li>Aseptically add 20 ml of LB + Amp in a 125 ml Erlenmeyer flask and heat to 42 &#176;C. For greater ministring production, prepare 500 ml of LB + Amp, preheated to 42 &#176;C.</li>
	<li>Transfer the resuspension into the preheated medium.</li>
	<li>Incubate at 42 &#176;C and 250 rpm until past log phase, indicated by A600 = 1.0. Do not leave 20 ml of culture at 42 &#176;C past 1 hr.</li>
	<li>Reduce the temperature to 37 &#176;C and leave the culture for an additional 90 min. For 500 ml, leave the culture for an additional induction period of 90 - 120 min and do not decrease the temperature.</li>
	<li>Reduce the temperature to 30 &#176;C and leave the culture shaking at 200 rpm for an additional temperature downshift period of 2 hr. For 500 ml, extend the temperature downshift to 4 hr or overnight.</li>
</ol>
4. Harvesting Ministrings
<ol>
	<li>Aseptically transfer the culture into sterile 50 ml centrifuge tubes. Centrifuge at 10,000 x g for 10 min and inspect for a pellet.</li>
	<li>Decant the supernatant into an appropriate biohazard waste container without disturbing the pellet. Repeat until all the cells have been collected.</li>
	<li>Retain the pellet for plasmid extraction with any commercial extraction and endotoxin removal kit or flash freeze in liquid nitrogen and store at -80 &#176;C for later extraction.</li>
</ol>

<ol>
	<li>Klinman, D. M., Ylt, A. K., Beaucaget, S. L., Conover, J., Kriegt, 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=CpG+motifs+present+in+bacterial+DNA+rapidly+induce+lymphocytes+to+secrete+interleukin+6,+interleukin+12,+and+interferon+&#947;.">CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon &#947;.</a> Proc Natl Acad Sci. 93, 2879-2883 (1996).</li><li>Hodges, B. L., Taylor, K. M., Joseph, M. F., Bourgeois, S. A., Scheule, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+transgene+expression+from+plasmid+DNA+gene+therapy+vectors+is+negatively+affected+by+CpG+dinucleotides.">Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides.</a> Mol Ther. 10 (2), 269-278 (2004).</li><li>Darquet, A. M., 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=Minicircle:+an+improved+DNA+molecule+for+in+vitro+and+in+vivo+gene+transfer.">Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer.</a> Gene Ther. 6 (2), 209-218 (1999).</li><li>Gracey Maniar, L. E., 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=Minicircle+DNA+vectors+achieve+sustained+expression+reflected+by+active+chromatin+and+transcriptional+level.">Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level.</a> Mol Ther. 21 (1), 131-138 (2012).</li><li>Catanese, D. J., Fogg, J. M., Ii, D. E. S., Gilbert, B. E., Zechiedrich, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercoiled+minivector+DNA+resists+shear+forces+associated+with+gene+therapy+delivery.">Supercoiled minivector DNA resists shear forces associated with gene therapy delivery.</a> Gene Ther. 19 (1), 94-100 (2011).</li><li>Hacein-bey-abina, 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=Insertional+oncogenesis+in+4+patients+after+retrovirus-mediated+gene+therapy+of+SCID-X1.">Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.</a> J Clin Invest. 118 (9), 3132-3142 (2008).</li><li>Nafissi, N., 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=DNA+ministrings:+highly+safe+and+effective+gene+delivery+vectors.">DNA ministrings: highly safe and effective gene delivery vectors.</a> Mol Ther Nucleic Acids. 3 (April), (2014).</li><li>Schakowski, 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=A+novel+minimal-size+vector+(MIDGE)+improves+transgene+expression+in+colon+carcinoma+cells+and+avoids+transfection+of+undesired+DNA.">A novel minimal-size vector (MIDGE) improves transgene expression in colon carcinoma cells and avoids transfection of undesired DNA.</a> Mol Ther. 3 (5 Pt 1), 793-800 (2001).</li><li>Wang, H. 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=A+novel+micro-linear+vector+for+in+vitro+and+in+vivo+gene+delivery+and+its+application+for+EBV+positive+tumors.">A novel micro-linear vector for in vitro and in vivo gene delivery and its application for EBV positive tumors.</a> PLoS One. 7 (10), (2012).</li><li>L&#243;pez-Fuertes, L., 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=DNA+vaccination+with+linear+minimalistic+(MIDGE)+vectors+confers+protection+against+Leishmania+major+infection+in+mice.">DNA vaccination with linear minimalistic (MIDGE) vectors confers protection against Leishmania major infection in mice.</a> Vaccine. 21 (3-4), 247-257 (2002).</li><li>Schakowski, 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=Minimal+size+MIDGE+vectors+improve+transgene+expression+in+vivo.">Minimal size MIDGE vectors improve transgene expression in vivo.</a> In Vivo. 21 (1), 17-23 (2007).</li><li>Hertwig, S., Klein, I., Lurz, R., Lanka, E., Appel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PY54,+a+linear+plasmid+prophage+of+Yersinia+enterocolitica+with+covalently+closed+ends.">PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends.</a> Mol Microbiol. 48, 989-1003 (2003).</li><li>Nafissi, N., Slavcev, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+an+in-vivo+linear+covalently+closed+DNA+vector+production+system.">Construction and characterization of an in-vivo linear covalently closed DNA vector production system.</a> Microb Cell Fact. 11 (1), 154 (2012).</li><li>Xu, Z. L., 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=Optimization+of+transcriptional+regulatory+elements+for+constructing+plasmid+vectors.">Optimization of transcriptional regulatory elements for constructing plasmid vectors.</a> Gene. 272 (1-2), 149-156 (2001).</li><li>Hoffmann, F., Rinas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+induced+by+recombinant+protein+production+in+Escherichia+coli.">Stress induced by recombinant protein production in Escherichia coli.</a> Adv Biochem Eng Biotechnol. 89, 73-92 (2004).</li><li>Sum, C., Chong, J. Y., Wettig, S., Slavcev, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+purification+of+linear+covalently+closed+deoxyribonucleic+acid+by+Q-anion+exchange+membrane+chromatography.">Separation and purification of linear covalently closed deoxyribonucleic acid by Q-anion exchange membrane chromatography.</a> J Chromatogr A. 1339, 214-218 (2014).</li><li>Hoffmann, F., Rinas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid+amplification+in+Escherichia+coli+after+temperature+upshift+is+impaired+by+induction+of+recombinant+protein+synthesis.">Plasmid amplification in Escherichia coli after temperature upshift is impaired by induction of recombinant protein synthesis.</a> Biotechnol Lett. 23, 1819-1825 (2001).</li><li>Nafissi, N., Sum, C. H., Wettig, S., Slavcev, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+one-step+heat-inducible+in+vivo+mini+DNA+vector+production+system.">Optimization of a one-step heat-inducible in vivo mini DNA vector production system.</a> PLoS One. 9 (2), (2014).</li></ol>

The authors declare they have no competing financial interests.

We describe here a simple system for the production of LCC DNA ministrings using a one-step heat-inducible protocol, which requires no special equipment aside from that which is used in typical bacterial growth and manipulation. DNA ministrings are torsion-free, stable linear DNA expression cassettes, devoid of prokaryotic genetic elements. They may be used in place of conventional methods for gene transfer, expression of reporter genes, as well as in assessments towards the efficiency of transgene expression in eukaryotic systems. DNA ministrings combine the biocompatibility and increased transfection efficiency benefits of &#34;mini&#34; vectors with the superior safety profile of linear DNA vectors. Our robust one-step in vivo production platform rapidly and easily produces DNA ministrings for any gene transfer application without the need for multiple costly in vitro processes.
There are several critical steps to ensure optimal ministring production (Figures 2 and 3). Heat induction is the most critical step to ministring production. Complete lack of ministring production is most likely due to lack of heat induction (cells retained at 30 &#176;C), where repression of protelomerase expression prevents conversion of precursor plasmid to LCC ministring. Following the heat induction protocol, expect a production efficiency of approximately 75%. Heat induction is optimal while cells are in log phase. Though there is no statistically significant difference in ministring PE when using cells in stationary phase (&#34;Overgrown&#34;, Figure 3), we did observe almost a 50% decrease in overall plasmid yield. Yields will depend on the plasmid extraction protocol used. For optimal ministring production, trigger heat induction while cells are in log phase. Poor ministring production will most likely be a result of prolonged temperature upshift or insufficient temperature downshift. Prolonged temperature upshift is discouraged as this activates the E. coli heat shock response15, which may inhibit protelomerase activity and interfere with plasmid stability. Therefore, timing the temperature downshift is another critical step for efficient ministring production. Without additional incubation time at 30 &#176;C, ministring production efficiency was found to be very poor (&#34;Early Removal&#34;, Figure 3). This may be attributed to the amount of time required for Tel expression and activity. The originating prophage PY54 encoding Tel protelomerase normally infects Yersinia, which optimally grows around 28 &#176;C12, indicating that 30 &#176;C may also be more optimal for Tel activity.
The DNA minivector production system utilizes an in vivo platform to generate high quality bacterial sequence-free DNA minivectors. Modifications to the protocol may include altering the growth media to optimize plasmid and protein production in order to promote cell growth during the growth phase (TB instead of LB), or increase ministring production and conversion efficiency. For larger culture volumes (200 ml and greater), temperature downshift and incubation at 30 &#176;C may be extended overnight without severe negative impact on ministring PE. We have included modifications in our protocol for 500 ml cultures as an example. Purification of DNA ministrings can be expedited through in vitro endonuclease digestion of the prokaryotic backbone. There are a number of endonuclease target sites (e.g., PI-SceI, PvuI, ScaI) available on the prokaryotic backbone of the precursor plasmid, which can be cut to expose open linear ends, allowing for in vitro degradation of the prokaryotic backbone. Isolation of ministrings from other vector species can also be achieved through anion exchange membrane chromatography16.
One current limitation associated with the LCC DNA vector production system is the need for purification of the DNA ministring from other LCC and CCC products. Although easily purified using standard plasmid isolation methods, the isolation step can still be a disadvantage in a large-scale setting. Separation of the ministring can be time-consuming using AGE if the ministring and the LCC prokaryotic backbone are too similar in size. Alternatively, anion exchange membrane chromatography may provide better separation of ministrings from CCC vector species16. Temperature upshift can be another potential limitation as high temperatures also activate the heat shock response in E. coli, which negatively affects recombinant protein yields17 and consequently reduce rates of plasmid to ministring conversion. We report elsewhere optimizations of the production platform to circumvent and/or mitigate such effects of heat shock18. We are also currently optimizing methods to eliminate or reduce LCC prokaryotic backbone contamination in vivo.
Chromosomal integration of the LCC DNA ministring disrupts the host chromosome and subjects the cell to apoptosis. Not only does this reduce potential negative consequences of insertional mutagenesis such as oncogenesis and silencing of adjacent genes, thereby improving its safety; the lethal effect can also serve as an ideal method for knock-in and gene replacement studies without the associated side effects and damage of integration. DNA ministring vectors can be applied towards the transfer and expression of genes for therapeutic proteins, RNA, antibodies, or antigens into a given target in vivo or replace a mal- or non- functional allele. The simple one-step heat-inducible in vivo production system allows DNA ministring vectors to be easily produced for clinical or industrial applications.

The goal is to produce scalable quantities of linear covalently closed (LCC) DNA minivectors using a simple and high efficiency one-step heat-inducible in vivo DNA ministring production system. DNA ministrings provide a safe and effective non-viral strategy to deliver DNA. They combine the safety of LCC vectors with the efficiency of DNA minicircles, and also offer a safer alternative to virus-derived vectors without compromising transfection efficiency.
In order for a transgene delivery system to be successful, the DNA vector must enter the target host cell and express the encoded transgene(s). There are several cellular barriers that need to be overcome in practice, particularly in mammalian systems. In order to avoid degradation by serum nucleases and immune detection by reticulo-endothelial system, the DNA vector must be bio- and immune-compatible with target system. Unprotected DNA is quickly digested by plasma nucleases and the plasmid membrane is composed of dense lipoprotein barriers so the DNA vector must be capable of rapidly crossing the plasma membrane of target cells. Once in the cell, vectors must traverse the cytoplasm and pass through the nuclear membrane to enter the nucleus for transgene expression. Non-viral gene delivery techniques focus on enhancement of tissue- and cell-targeting. However, the use of conventional plasmids with these techniques reduces transfection efficiency due to the presence of immunogenic bacterial sequences, which rapidly silences gene expression1,2. Conventional plasmids typically carry antibiotic resistance genes for maintenance in prokaryotic systems. However, these may produce potential adverse effects in human hosts or impart resistance to naturally occurring host flora via horizontal gene transfer effects. Conventional plasmids also contain dinucleotide CpG motifs2, which can trigger an unwanted immunostimulatory response, potentially reducing or silencing transgene expression.
As they are solely comprised of the eukaryotic expression cassette, DNA minivectors, such as DNA minicircles3, are a better alternative for gene delivery as they exhibit improved extracellular and intracellular bioavailability and improved gene expression due to their reduced size and absence of immunostimulatory prokaryotic elements4. The reduced vector size fares better with respect to resistance to shear forces associated with in vivo administration to a target site5. The higher copy number of the vector per unit mass requires less transfection reagent, thus decreasing toxicity. However, in the event of random vector integration into a host chromosome, circularly covalently closed (CCC) vectors, including both DNA minicircles and conventional plasmids, impart molecular continuity and therefore may lead to insertional mutagenesis, which can have devastating consequences6. Comparatively, integration of a linear DNA vector disrupts the chromosome and initiates cell death pathways, thereby removing the mutant cell from the proliferating cell population and preventing insertional mutagenesis7. Minimalistic immunologically defined gene expression (MIDGE) vectors8 and micro-linear vectors9 (MiLV) are LCC DNA vectors developed in vitro. MIDGE vectors have exhibited up to a 17-fold improved transgene expression in vivo compared to conventional plasmid DNA vectors11, and have demonstrated promising results in vaccine10 and cancer8 gene therapy. In addition, due to the torsion-free structure of the LCC minivector, less transfection reagent is required in comparison to the CCC supercoiled counterpart, thus reducing toxicity7.
Our enhanced LCC DNA vectors, DNA ministrings, have demonstrated superior expression efficiency and bioavailability7. Furthermore, DNA ministrings are produced on a one-step heat-inducible in vivo production system, an expedient and cost-effective alternative to MIDGE and MiLV LCC vectors, which require multiple steps in vitro. The DNA ministring production system to be demonstrated is a simple heat-inducible process performed in vivo, making it both cost-effective and easily scalable. This system exploits the Yersinia enterocolitica bacteriophage PY54-derived Tel/pal protelomerase recombination system12 to separate the minimal eukaryotic expression cassette from the prokaryotic plasmid backbone. We have engineered Escherichia coli cells (W3NN) to express Tel protelomerase under the control of the heat-inducible bacteriophage &#955; promoter, cI[Ts]85713. Upon expression, Tel protelomerase acts on pal target sites present within &#34;Super Sequence&#34; (SS) sites located on the precursor plasmid to yield LCC products, from which the DNA ministring can then be purified (Figure 1). The SS sites on the DNA ministring precursor plasmid also encode target sites for other recombinases including Cre recombinase (lox), TelN protelomerase (telRL) and Flp recombinase (FRT), thereby facilitating the production of isogenic LCC and CCC minivectors from one precursor plasmid. In addition, each SS site is flanked on both sides by SV40 enhancer (SV40e) sequences, which serve to improve nuclear translocation14. We have demonstrated elsewhere that successive addition of SV40e progressively confers corresponding increases in transfection efficiency7. The precursor plasmid (pDNA MiniString or pDMS) contains a polylinker (Figure 1) to facilitate insertion of any desired gene of interest in transcriptional fusion with the green fluorescent reporter (GFP).
The DNA minivector technology may be used in place of conventional methods for gene transfer, expression of reporter genes, and assessments towards the efficiency of transgene expression in eukaryotic systems. DNA ministrings combine the biocompatibility and increased transfection efficiency benefits of &#34;mini&#34; vectors with the superior safety profile of linear DNA vectors. Our robust one-step production platform rapidly and easily produces DNA ministrings for any gene transfer application and offers a higher safety profile.

<table><tbody><tr><td>&lt;em&gt;E. coli&lt;/em&gt; W3NN</td><td>Mediphage</td><td></td><td>strain for ministring processing: Nafissi &amp;amp; Slavcev, 2012</td></tr><tr><td>pDMS.IRES.GFP</td><td>Mediphage</td><td></td><td>parent plasmid for ministring processing: also referred to as pDNA Ministring (pDMS)</td></tr><tr><td>BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani)</td><td>Fisher Scientific</td><td>244620</td><td></td></tr><tr><td>Fisher BioReagents Terrific Broth</td><td>Fisher Scientific</td><td>BP2468500</td><td></td></tr><tr><td>BD Bacto Dehydrated Agar</td><td>Fisher Scientific</td><td>214010</td><td></td></tr><tr><td>Ampicilin</td><td>MP Biomedicals LCC</td><td>2190147</td><td></td></tr><tr><td>Ultrapure Milipore Water</td><td>Fisher Scientific&amp;nbsp;</td><td>Z00Q0V0US</td><td>Model: Mili-Q Advantage A10 Water Purification System</td></tr><tr><td>Surgical Gloves</td><td>VWR</td><td>(S) 82026-424, (M)82026-426, (L) 82026-428, (XL) 82026-430&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Fisherbrand Petri Dishes with Clear Lid Raised Ridge (100&amp;nbsp;x&amp;nbsp;15&amp;nbsp;mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>30 ml KIMAX Test Tubes</td><td>VWR</td><td>89001-448</td><td></td></tr><tr><td>Spectrophotometer Cuvette</td><td>Sarstedt</td><td>67.74</td><td></td></tr><tr><td>Sterile 15 ml Falcon Tubes</td><td>&amp;nbsp;BD Falcon</td><td>62406-200,</td><td></td></tr><tr><td>Sterile 50 ml Centrifuge Tubes</td><td>&amp;nbsp;FroggaBio</td><td>2930-S0</td><td></td></tr><tr><td>Falcon Disposable Polystyrene Serological Pipets 10 ml</td><td>BD Falcon</td><td>13-675-20</td><td></td></tr><tr><td>Falcon Disposable Polystyrene Serological Pipets 25 ml</td><td>BD Falcon</td><td>13-668-2&amp;nbsp;</td><td></td></tr><tr><td>Micropipettor (100-1,000&amp;nbsp;&amp;mu;l)</td><td>FroggaBio</td><td>C1000-1</td><td></td></tr><tr><td>Micropipettor (20-200&amp;nbsp;&amp;mu;l)</td><td>FroggaBio</td><td>C200-1</td><td></td></tr><tr><td>Sterile Pipette Tips (100-1,250&amp;nbsp;&amp;mu;l)</td><td>VWR</td><td>89079-472&amp;nbsp;</td><td></td></tr><tr><td>Sterile Pipette Tips (1-200&amp;nbsp;&amp;mu;l)</td><td>VWR</td><td>89079-460</td><td></td></tr><tr><td>Fisher Scientific Economy Burner</td><td>Fisher Scientific</td><td>03-917Q</td><td></td></tr><tr><td>Incubator Shaker</td><td>New Brunswick Scientific&amp;nbsp;</td><td>M1352-00000</td><td></td></tr><tr><td>Sterile Erlenmeyer Flask (125 ml)</td><td>Pyrex (Sigma Aldrich)</td><td>CLS4980125 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Sterile Erlenmeyer Flask (250 ml)</td><td>Pyrex (Sigma Aldrich)</td><td>CLS4980250 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Sterile Erlenmeyer Flask (500 ml)</td><td>Pyrex (Sigma Aldrich)</td><td>CLS4980500 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>335901</td><td>Model: Genesys 10 Vis</td></tr><tr><td>Floor Centrifuge</td><td>Beckman Coulter&amp;nbsp;</td><td>369001</td><td>Model: Avanti J-E</td></tr><tr><td>Benchtop Centrifuge</td><td>Beckman</td><td>362114</td><td>Model: GS-6R</td></tr><tr><td>Fisher Scientific Analog Vortex Mixer&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>02-215-365</td><td></td></tr><tr><td>1 kb DNA Ladder</td><td>New England BioLabs</td><td>N3232S</td><td></td></tr></tbody></table>

production of double-stranded dna ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

After DNA extraction, residual precursor plasmid, LCC prokaryotic backbone, and the DNA ministring will be recovered. Overall plasmid yield and purity can be assessed using a spectrophotometer. Purity is estimated using the ratio of the absorbance at 260 nm and 280 nm (A260/A280) where a ratio of 1.9-2.0 is optimal. All LCC and CCC products can be separated through agarose gel electrophoresis (AGE) (Figure 2). AGE visualizes the results of ministring production after heat induction, prior to ministring purification. A qualitative assessment of ministring production can be made at this stage by comparing band intensity of the ministring (2.4 kb) to parent plasmid (5.6 kb) (Figure 2). Ministring DNA can be recovered via standard plasmid gel extraction protocols following AGE. Figure 3 summarizes ministring production efficiencies under various conditions likely to occur while carrying out the protocol. Ministring production efficiency was determined based on DNA band intensity using the manufacturer&#39;s analysis program. Any similar densitometry program can be used to analyze the results of AGE.
<img alt="Figure 1" src="/files/ftp_upload/53177/53177fig1highres.jpg" width="700" /> 
Figure 1: Overview of ministring production. (A) The parent vector contains a polylinker in transcriptional fusion with a reporter gene, framed by the two SS sites. The backbone encodes beta-lactamase for ampicillin resistance and also contains several cut sites unique to the backbone for in vitro digestion. (B) Overview of the main protocol. Steps 2) Growth Phase and 3) Ministring Production are represented in a diagram to provide an overall schematic of the steps involved, highlighting the simplicity of the protocol. (C) Ministring induction. Heat induction inactivates the temperature sensitive repressor, allowing tel expression followed by Tel activity on SS sites in the parent plasmids. <a href="https://www.jove.com/files/ftp_upload/53177/53177fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53177/53177fig2highres.jpg" width="350" /> 
Figure 2: Ministring production after deviations from protocol. 1: Control, 2: Uninduced, 3: Overgrown, 4: Overinduced, 5: Early Removal. DNA ministring is approximately 2.4 kb, LCC backbone is 3 kb, and pNN9 parent plasmid is 5.6 kb. Band intensity of ministring and LCC backbone bands drop with deviations from protocol, indicating lower ministring yield. From left to right: 1 kb ladder from New England Biolabs; Control, following protocol; Uninduced, no temperature upshift; Overgrown, temperature upshift during stationary phase; Overinduced, extended temperature upshift; Early removal, no temperature downshift. Plasmids were extracted using the Omega-Biotek E.Z.N.A. Plasmid Mini Kit. <a href="https://www.jove.com/files/ftp_upload/53177/53177fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53177/53177fig3highres.jpg" width="550" /> 
Figure 3: Effects of deviations from protocol on ministring production efficiency (PE). Results are the mean of 3 trials. PE is estimated from relative band intensity measured using AlphaImager HP after AGE. The deviations occur at the following steps: Control, following protocol; Uninduced, no temperature upshift at step 2; Overgrown, temperature upshift began during stationary phase at step 1.6; Overinduced, extended temperature upshift at step 2.3; Early removal, cells removed and extracted without temperature downshift at step 2.3. <a href="https://www.jove.com/files/ftp_upload/53177/53177fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production of Double-stranded DNA Ministrings at JoVE.com

Production of Double-stranded DNA Ministrings

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

production-of-double-stranded-dna-ministrings

Research

JoVE Journal

Biology

10.3K Views.  University of Waterloo. The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings. 

Video: Production of Double-stranded DNA Ministrings

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

Genetics

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

Biochemistry

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single strands, each requiring distinct modes of replication: continuous (leading) and discontinuous (lagging). Understanding the interactions of chromatin-associated proteins with replicating DNA strands in vivo is crucial for elucidating the mechanisms of chromatin assembly and DNA repair coupled to DNA replication. This protocol presents the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method, designed to quantify relative protein levels on nascent leading and lagging DNA strands at replication forks. The eSPAN procedure starts with chromatin immunoprecipitation (ChIP, in yeast) or cleavage under targets and tagmentation (CUT&amp;Tag; in mammalian cells) of a protein of interest, followed by enrichment of the protein-associated nascent DNA by bromodeoxyuridine (BrdU) immunoprecipitation. Strand-specific next-generation sequencing is applied to isolated ssDNA. This technique can be used to determine whether a protein is enriched at leading or lagging replication forks. The eSPAN provides genome-wide strand-preference of chromatin-associated proteins, including histones at replication forks.

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single ...

The DNA Replication Fork

An organism&#8217;s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.&#160;&#160; Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication forks, one in each direction leading away from the location of the initial opening.&#160; In organisms with large genomes, the replication of DNA is not done from a single point of origin but in many distinct, localized replication forks.&#160;
The unhindered progression of the replication fork is necessary for complete DNA replication and genome stability; however, the replication fork is often stalled by internal or external factors that can slow or stop its progression, resulting in replication stress. Replication stress causes genomic instability, which is a hallmark of diseases like cancer. Genomic instability is characterized by genomic alterations and increased frequency of harmful mutations. The movement of the replication fork can stop due to several reasons. For example, the drug hydroxyurea depletes the pool of nucleotides available for incorporation into the daughter strand, stalling the replication fork. Other problems that may hinder the progression of the replication fork include DNA lesions, a collision between a replication fork and a transcription complex, and defects in the enzymes involved in DNA replication.
The cell has a variety of repair mechanisms to reinitiate the stalled replication fork.&#160; S-phase checkpoints do not allow cells to begin mitosis until DNA repair is complete. Additionally, fork repriming can restart DNA synthesis by bypassing a DNA lesion or block. Despite these robust mechanisms, sometimes stalled forks cannot be reinitiated, which leads to the collapse of a fork, halting DNA replication.

An organism&#8217;s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped ...

Education

JoVE Core

Molecular Biology

Unit 1

DNA Replication

KEY TERMS AND CONCEPTS

Fixing Double-strand Breaks

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages both copies of genetic information but also disrupts the continuity of DNA, making the chromosome fragile.
In a cell, there are an estimated ten double-strand breaks (DSBs) per day. The primary source of damage is metabolic by-products such as Reactive Oxygen Species and environmental factors such as ionizing radiations. Although less common, malfunctioning nuclear enzymes can also cause DSBs. Failure of enzymes like type II topoisomerases, which cut both strands of DNA and rejoin them while disentangling chromosomes, can inadvertently result in DSBs. Mechanical stress on the DNA duplex can also lead to DSBs. In prokaryotes, prolonged desiccation strains DNA, causing DSBs.
Of the two mechanisms for DNA repair, homologous recombination depends on a sister chromatid being nearby, which happens during the S and G2 phases. Due to this restriction, in the absence of a homology donor, cells have to resort to Nonhomologous end joining (NHEJ), even though it is much less accurate. It has been hypothesized that the reason higher eukaryotes can afford to preferentially utilize NHEJ for DSB repairs is that they have abundant non-coding DNA, which permits nucleotide substitutions, deletions or additions without grievous consequences.

Double-strand Breaks DSB &amp; Nonhomologous End Joining NHEJ

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the ...

DNA Repair and Recombination

Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell&#39;s circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
Many Proteins Work Together to Replicate the Chromosome
Replication is coordinated and carried out by a host of specialized proteins. Topoisomerase breaks one side of the double-stranded DNA phosphate-sugar backbone, allowing the DNA helix to unwind more rapidly, while helicase breaks the bonds between base pairs at the fork, separating the DNA into two template strands. Proteins that bind single-stranded DNA molecules stabilize the strands as the replication fork travels along the chromosome. DNA can only be synthesized in the 5&#39; to 3&#39; direction, so one strand of the template&#8212;the leading strand&#8212;is elongated continuously, while the other strand&#8212;the lagging strand&#8212;is synthesized in shorter pieces of 1,000&#8211;2,000 base pairs called Okazaki fragments.
Multiple Polymerases Take Part in Elongation
Much of the research to understand prokaryotic DNA replication has been performed in the bacterium Escherichia coli, a commonly-used model organism. E. coli has five DNA polymerases: Pol I, II, III, IV, and V. Pol III is responsible for the majority of DNA replication. It can polymerize about 1,000 base pairs per second. This astonishing pace allows the machinery present at the two replication forks to duplicate the E. coli chromosome&#8212;4.6 million base pairs&#8212;in roughly 40 minutes. DNA polymerase I is also well-characterized. Its primary role is to process the RNA primers on the lagging strand.
When Division Outpaces Duplication
Under favorable growth conditions, E. coli can divide every 20 minutes, about half the amount of time that it takes to replicate the genome. But how is this possible when both daughter cells must have their own DNA? Scientists found that the bacteria can begin another round of DNA replication from the origin of replication before the first round is complete; this means that daughter cells receive a chromosome that is already in the process of being copied and are prepared to divide again very quickly.

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the ...

Cell Biology

DNA Replication and Repair

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the likelihood of an identical or similar DNA sequence on a sister chromatid is high. Thus, the timing of repair prevents recombination between non-identical sequences. This is a critical feature of HR, particularly during the recombination of parental DNA sequences in an offspring, where faulty HR can lead to loss of the entire gene and the surrounding chromosomal region.
The accurate repair ensured by HR has been applied in gene-editing techniques. HR is the earliest method that has been used to edit genomes in living cells. The CRISPR-Cas9 system is used to create targeted double-strand breaks to correct disease-causing mutations in the genome. The isolated fragments are taken up by cells, where they can recombine with cellular DNA and replace the targeted region of the genome. HR mechanisms govern the repair of the breaks and their accurate recombination with the cellular genome. To help the HR proteins localize precisely at double-strand breaks, Cas9 proteins are fused with HR effector proteins that can recruit repair proteins at the damaged sites. Studies have shown that fusing Cas9 with proteins such as CtIP, Rad52, and Mre11 can increase HR events in the cell by two-folds while discouraging Non-homologous end joining. Such applications of HR in genome editing can revolutionize gene therapy and provide treatment for genetic diseases that are currently considered incurable.

Homologous Recombination and Strand Invasion

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of ...

The Replisome

DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the &#8220;Trombone model&#8221; was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the synthesis of an Okazaki fragment and finally dissolves when synthesis of the fragment ends. The DNA polymerase then attaches to another primer and the whole cycle, from loop formation to collapse, is repeated. The replisome allows all of the activities to be coordinated as a replication machinery complex.
Replisome components
Helicases unwind and separate double-stranded DNA.&#160; These enzymes are present as a single hexamer ring in prokaryotes and as a double hexamer ring in eukaryotes. Eukaryotic helicases depend on additional proteins, Cdc45 and GINS, to function.&#160; Single-stranded DNA binding (SSB) proteins keep the DNA strands from reannealing.&#160; In prokaryotes, SSBs consist of a single subunit, whereas in eukaryotes, they are a heterotrimeric protein known as replication protein A (RPA).
Primase adds an RNA primer to where DNA synthesis will originate.&#160; In prokaryotes, primase is present as a single subunit enzyme called DnaG, which synthesizes an RNA primer of around 12 nucleotides.&#160; In eukaryotes, a multisubunit enzyme, DNA polymerase-&#945; primase, synthesizes an RNA-DNA hybrid primer of around 25 nucleotides. In addition to polymerase-&#945;, replicative polymerase will extend the newly synthesized DNA. While a single type of replicative polymerase, DNA polymerase III, is present in prokaryotes, two different types of replicative polymerases, Pol &#949; and Pol &#948;, are present in eukaryotes, for leading strand and lagging strand synthesis, respectively.&#160;
Sliding Clamp proteins keep the polymerases attached to the DNA template. &#160;&#946;-clamp, a homodimeric protein, acts as the clamp in prokaryotes. In eukaryotes, the same task is performed by proliferating cell nuclear antigen (PCNA), a homotrimeric protein. The central pore of the sliding clamp is positively charged, which enables it to have electrostatic interactions with the negatively charged phosphate backbone of the DNA. The clamp is attached to the DNA by a Clamp Loader. These pentameric proteins belong to the AAA+ class of ATPases. The eukaryotic clamp loader is known as replication factor C, while the prokaryotic E. coli clamp loader is known as the &#947; complex; however, the clamp proteins and clamp loaders are thought to be evolutionary homologs unlike many other components of the replisome.

The Replisome Components and the Trombone Model

DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this ...

DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.&#160; After replication, each double-stranded DNA includes one parental or &#8220;old&#8221; strand and one &#8220;new&#8221; strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
DNA replication uses a large number of proteins and enzymes. The enzyme helicase separates the two strands of DNA. As helicase moves along the DNA, it separates the two strands to form a Y-shaped structure known as the replication fork. Following this, the enzyme primase adds short stretches of RNA known as primers to initiate the synthesis of DNA by DNA polymerase, the enzyme responsible for DNA synthesis. In bacteria, three main types of DNA polymerases are known: DNA pol I, DNA pol II, and DNA pol III. DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair. DNA pol III adds deoxyribonucleotides, each complementary to a nucleotide on the template strand, one by one to the 3&#8217;-OH group of the growing DNA chain. DNA polymerase III can only extend in the 5&#8217; to 3&#8217; direction. The addition of these nucleotides requires energy. This energy is present in the bonds of three phosphate groups attached to each nucleotide, similar to how energy is stored in the phosphate bonds of adenosine triphosphate (ATP). When the bond between the phosphates is broken and diphosphate is released, the energy released allows for the formation of a covalent phosphodiester bond by dehydration synthesis.
The DNA double helix is antiparallel; that is, one strand is oriented in the 5&#8217; to 3&#8217; direction and the other is oriented in the 3&#8217; to 5&#8217; direction. During replication, one strand, which is complementary to the 3&#8217; to 5&#8217; parental DNA strand, is synthesized continuously toward the replication fork because polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5&#8217; to 3&#8217; parental DNA, grows away from the replication fork, so the polymerase must move back toward the replication fork to begin adding bases to a new primer, again in the direction away from the replication fork. It does so until it bumps into the previously synthesized strand, and then it moves back again. These steps produce small DNA sequence fragments known as Okazaki fragments, each separated by RNA primer. The strand with the Okazaki fragments is known as the lagging strand, and its synthesis is said to be discontinuous.
After the synthesis by DNA polymerase III, the primers are removed by the exonuclease activity of DNA polymerase I, and the gaps are filled in. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of a covalent phosphodiester linkage between the 3&#8217;-OH end of one DNA fragment and the 5&#8217; phosphate end of the other fragment, stabilizing the sugar-phosphate backbone of the DNA molecule.
Replication in Eukaryotes
Eukaryotic genomes are much more complex and larger than prokaryotic genomes and are typically composed of multiple linear chromosomes. The human genome, for example, has 3 billion base pairs per haploid set of chromosomes, and 6 billion base pairs are inserted during replication. There are multiple origins of replication on each eukaryotic chromosome; the human genome has 30,000 to 50,000 origins of replication. The rate of replication is approximately 100 nucleotides per second&#8212;10 times slower than prokaryotic replication.
The essential steps of replication in eukaryotes are the same as in prokaryotes. Before replication can start, the DNA has to be made available as a template. Eukaryotic DNA is highly supercoiled and packaged, which is facilitated by many proteins, including histones. Following initiation of replication, in a process similar to that found in prokaryotes, elongation is facilitated by eukaryotic DNA polymerases. The leading strand is continuously synthesized by the eukaryotic polymerase enzyme pol &#948;, while the lagging strand is synthesized by pol &#949;. The enzyme ribonuclease H (RNase H), instead of a DNA polymerase as in bacteria, removes the RNA primer, which is then replaced with DNA nucleotides. The gaps that remain are sealed by DNA ligase.
As in prokaryotes, the eukaryotic DNA polymerase can add nucleotides only in the 5&#8217; to 3&#8217; direction. In the leading strand, synthesis continues until it reaches either the end of the chromosome or another replication fork progressing in the opposite direction. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no place to make a primer for the DNA fragment to be copied at the end of the chromosome. These ends thus remain unpaired, and, over time, they may get progressively shorter as cells continue to divide.
This text is adapted from <a href="https://openstax.org/books/microbiology/pages/11-2-dna-replication">Openstax, Microbiology, Chapter 11.2: DNA Replication</a>.

DNA Replication: Semiconservative, 5'-3' DNA Synthesis

DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary ...