We thank C.C.R. laboratory members for comments and S. Moskowitz for figure preparation. This work was supported by National Institutes of Health Grants F32GM101761 (A.J.H.) and GM54397 (C.C.R.).

NOTE: Follow all institutional regulations regarding the safe use and disposal of radioactive material, including but not limited to, usage of personal protective equipment, such as gloves, safety glasses, lab coat, and appropriate acrylic shields.
NOTE: The standard buffer consists of 40 mM HEPES-KOH, pH 7.5, 50 mM potassium glutamate, 5 mM DTT, 0.1 mM EDTA (this buffer is pre-made at a 5x concentration) and 0.3 mM of each dNTP. T7 replication proteins are purified as described8,9,10. Single-stranded DNA containing a single T7 primase recognition site (5&#39;-GGGTC-3&#39;, 5&#39;-GTGTC-3&#39;, 5&#39;-TGGTC-3&#39;) can be purchased from a variety of DNA synthesis companies (the length of the ssDNA may depend on the enzyme of choice, for T7 primase, a pentamer is the minimum template length to synthesize a full-length primer11, as the cryptic C is essential, however, if the primer is to be extended by polymerase, additional nucleotides must be present at the 3&#39; end of the template. Reactions are initiated by the addition of a final concentration of 10 mM MgCl2 and incubated at 25 &#176;C. Manual sampling works well for time-points of 5 seconds or greater. If time-points shorter than that are required, it is recommended to use a rapid-quench flow instrument to obtain accurate data.
1. Multiple-turnover Primer Synthesis Reaction by Manual Sampling
<ol>
	<li>Before starting the experiment, aliquot 2 &#181;L of formamide dye buffer (93% (v/v) formamide, 50 mM EDTA, 0.01% xylene cyanol and 0.01% bromophenol blue) into 8 1.5 mL polypropylene tubes. In general, the number of tubes is equal to the number of time-points analyzed.</li>
	<li>Prepare a 20 &#181;L mixture consisting 1x buffer, 0.1 mM ATP and CTP, 0.25 mCi/mL [&#945;-32P] CTP, 0.1 &#181;M ssDNA template, and 0.1 &#181;M gp4 (expressed as hexamer concentration &#8212; 0.6 &#181;M monomer).</li>
	<li>Incubate the mixture at room temperature for 5 min.</li>
	<li>Remove 2 &#181;L of the mixture using a pipettor and mix with 2 &#181;L formamide dye buffer in a 1.5 mL tube prepared in step 1.1 for the no reaction control (time zero).</li>
	<li>Add 2 &#181;L of 0.1 M MgCl2 to reaction mixture (to a final concentration of 10 mM).</li>
	<li>Manually withdraw 2 &#181;L samples of the reaction mixtures using a pipettor at 10 s intervals and mix with formamide dye predispensed in 1.5 mL tubes prepared in step 1.1.</li>
	<li>Heat samples at 95 &#176;C for 5 min in a heat block.</li>
	<li>Load aliquots of the samples onto a denaturing gel. The small tetraribonucleotide primer products synthesized by the T7 primase are resolved well on a 0.4 mm thick 25% acrylamide:bisacrylamide (19:1), 3 M urea, 1x TBE (100 mM Tris-borate, 1 mM EDTA).</li>
	<li>Place entire gel on chromatography paper and cover with plastic wrap. Dry gel using a gel drier.</li>
	<li>Prepare a dilution series of the remainder of the reaction mixture and spot the same volume loaded on the gel onto chromatography paper, i.e., spot 4 &#181;L of the dilution if 4 &#181;L of sample were loaded. 
	NOTE: This will serve as the standard curve to determine the concentration of products formed during the reaction. For example, five-fold dilutions using TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) work well, spanning a range of 1:25 to 1:15,625 (52-56), but the dilutions should be adjusted depending on the level of activity.</li>
	<li>Expose dry gel and standard using a phosphorimager screen. Quantify the radioactivity in the dilution series and construct a standard curve as described 8,12 
	NOTE: Inclusion of T7 DNA polymerase to the reaction mixture allows one to examine the usage of tetraribonucleotide primers by this enzyme. Optimally, the polymerase concentration should be saturating to simplify the interpretation of the data. A polymerase concentration of 0.4 &#181;M or higher gives good results. Additionally, the effect of other proteins, such as single-stranded DNA binding proteins, on either primer synthesis or lagging-strand initiation can be examined in this manner.</li>
</ol>
2. Multiple-turnover Primer Synthesis Reaction Using a Rapid-quench Instrument
<ol>
	<li>Preparation of reaction mixtures
	<ol>
		<li>Prepare 400 &#181;L of solution A, containing 1x buffer, 0.2 &#181;M gp4 hexamer, 0.2 &#181;M ssDNA template, 0.6 mM dNTPs, 0.2 mM ATP and CTP (including 0.5 mCi/mL [&#945;-32P] CTP.</li>
		<li>Prepare 400 &#181;L of solution B, containing 1x buffer and 20 mM MgCl2.</li>
	</ol>
	</li>
	<li>Operation of rapid-quench flow instrument
	<ol>
		<li>Turn on rapid-quench flow instrument; after the main menu appears, type 7 on the instrument keyboard to move the syringe driver to the home position.</li>
		<li>Open buffer syringe position to &#34;LOAD&#34;.</li>
	</ol>
	</li>
	<li>Loading buffer syringes
	<ol>
		<li>Attach a 10 mL syringe containing buffer to the drive syringe port. Change syringe knob position to &#34;LOAD&#34;.</li>
		<li>Using 10 mL syringes, fill instrument buffer reservoir A and B with 10 mM Tris, pH 7.5, 1 mM EDTA and fill syringe C with quench solution (250 mM EDTA, and 0.2% SDS). Remove air bubbles by pushing plungers in and out.</li>
		<li>Change knob positions to &#34;FIRE&#34;. Type 2 to adjust position of the syringe driver bar. Press &#34;ADJUST DOWN&#34; button to lower the syringe until buffer comes out the exit loop: (7-goes down continuously; 8-large step down; 9-small step down). Type &#34;ESC&#34; to return to the main menu.</li>
	</ol>
	</li>
	<li>Wash tubing
	<ol>
		<li>Attach exit line to vacuum. Change sample knobs to &#34;FLUSH&#34;. Close buffer syringe by setting the knobs to the horizontal position. Turn on vacuum and dip the 2 flush loops in H2O for 10 s, then dip in MeOH for 10 s. Dry loops by sucking air for ~15 s, or until dry.</li>
	</ol>
	</li>
	<li>Loading samples
	<ol>
		<li>Change sample knob position to &#34;LOAD&#34;. Place solution A into 1 mL syringe. Plug syringe into one of the sample load ports. Repeat for solution B, and place in opposite sample load port.</li>
	</ol>
	</li>
	<li>Collection of time-points
	<ol>
		<li>On main menu, type 1 for quench-flow run. Enter reaction time (s) and press, &#34;ENTER&#34;. The reaction loop number to use will be displayed. Switch to required reaction loop. Repeat wash procedure in step 2.2.4.</li>
		<li>Turn sample knobs to &#34;LOAD&#34;. Load reaction mixes in sample loops until just outside the central mixing compartment. Turn all knobs to &#34;FIRE&#34;. Confirm that all knobs are in the correct position.</li>
		<li>Place 1.5 mL tube onto end of exit loop. Press &#34;GO&#34; to run the reaction. Repeat wash procedure in step 2.2.4. Reload buffer syringes A and B until they hit the driver.</li>
		<li>Repeat steps 2.2.6.2-2.2.6.3 for each desired time point. Exception: Don&#39;t load solution containing the start reagent (i.e., MgCl2 in this case) when quenching the zero time-point control.</li>
		<li>When finished collecting samples, enter &#34;ESC&#34; to return to main menu. Press &#34;7&#34; to return buffer syringe driver to its home position.</li>
	</ol>
	</li>
	<li>Instrument clean-up
	<ol>
		<li>Remove reaction mixture syringes. Turn sample loading knob to &#34;LOAD&#34; position. Under vacuum, wash each sample loop with 10 mL 0.2 N NaOH, followed by 10 mL 0.3 N H3PO4 and 10 mL MeOH.</li>
		<li>Turn syringe knobs to &#34;LOAD&#34; position. Press down on all 3 syringe plungers to retrieve reaction and quench buffers. Wash syringes with 5 mL H2O at least twice.</li>
		<li>Fill syringes again with 5 mL H2O and turn knobs to &#34;FIRE&#34; position. Change knob positions and turn on vacuum to remove H2O. Flush as in step 2.2.4. Turn off instrument. 
		NOTE: Products are analyzed as described in steps 1.7-1.12. In addition, T7 DNA polymerase and other replication proteins can be added in the reaction mixture to analyze their effect on primer synthesis or extension.</li>
	</ol>
	</li>
</ol>
3. Single-turnover Primer Synthesis Reaction
NOTE: Single-turnover primer synthesis/extension assays are carried out similarly to multiple-turnover reactions, with a few major differences: these assays follow the conversion of radiolabeled ATP into primer or extension products, however the concentration of substrates and proteins are considerably higher. In addition, in order to attain millisecond resolution to allow analysis of the primer synthesis reaction, one must utilize a rapid-quench flow machine.
<ol>
	<li>Preparation of reaction mixtures.
	<ol>
		<li>Prepare 400 &#181;L of solution A, containing 1x buffer, 3 &#181;M gp4 hexamer, 6 &#181;M ssDNA template, 0.6 mM dNTPs, 1 mM CTP, and 2 nM [&#947;-32P] ATP. Prepare 400 &#181;L of solution B, containing 1x buffer and 20 mM MgCl2.</li>
	</ol>
	</li>
	<li>Rapid-quench flow operation
	<ol>
		<li>Carry out reactions as described in step 2.2. If desired, add T7 DNA polymerase or T7 single-stranded-DNA binding protein at a concentration of 15 &#181;M and 20 &#181;M, respectively. In this case, the extension products accumulate at a time regime that allows for manual sampling of the reaction.</li>
	</ol>
	</li>
</ol>
4. Data Analysis
<ol>
	<li>Fit product progress curves to non-linear least-squares models using commercially available software capable of least-squares fits and/or numerical integration. The details of the operation will vary with the software employed, but below are generalized forms of equations used for fitting the data.</li>
	<li>Determine the rate of product formation of multiple turnover primer synthesis reactions by fitting to progress curves to a linear equation. If there is significant curvature, use the initial rate method13,14 by fitting the data to a single-exponential function: y=A(e-kt ), where y is the product concentration, A is the reaction amplitude, k is the observed rate constant, t is time, in seconds. The slope of the tangent line at time=0 is the initial rate. 
	NOTE: Fit pre-steady state primer synthesis reactions to a linear equation or to the pre-steady state burst equation y=A (e-kburstt )+ratess t, where A is the reaction amplitude, kburst is the observed rate constant for the pre-steady state burst, ratess is the steady-state ratess =kcat [Enzyme]), t is time, in seconds. Fit single-turnover primer synthesis reactions progress curves to a single-exponential function y=A(e-kt). In the case of gp4, fit the data by numerical integration15, to a model with three NTP condensation steps, where each intermediate is in equilibrium with the enzyme, using commercially available software.</li>
</ol>

<ol>
	<li>Kornberg, A., Baker, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DNA replication. , (1992).</li><li>Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motors,+switches,+and+contacts+in+the+replisome.">Motors, switches, and contacts in the replisome.</a> Annu Rev Biochem. 78, 205-243 (2009).</li><li>Mendelman, L. V., Notarnicola, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+bacteriophage+T7+gene+4+proteins+in+providing+primase+and+helicase+functions+in+vivo.">Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo.</a> Proc Natl Acad Sci U S A. 89 (22), 10638-10642 (1992).</li><li>Qimron, U., Lee, S. J., Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+initiation+and+extension+by+T7+DNA+primase.">Primer initiation and extension by T7 DNA primase.</a> EMBO J. 25 (10), 2199-2208 (2006).</li><li>Kato, M., Ito, T., Wagner, G., Richardson, C. C., Ellenberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+architecture+of+the+bacteriophage+T7+primase+couples+RNA+primer+synthesis+to+DNA+synthesis.">Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis.</a> Mol Cell. 11 (5), 1349-1360 (2003).</li><li>Kusakabe, T., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+4+DNA+primase+of+bacteriophage+T7+mediates+the+annealing+and+extension+of+ribo-oligonucleotides+at+primase+recognition+sites.">Gene 4 DNA primase of bacteriophage T7 mediates the annealing and extension of ribo-oligonucleotides at primase recognition sites.</a> J Biol Chem. 272 (19), 12446-12453 (1997).</li><li>Lee, S. J., Zhu, B., Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+sequence-specific+template+binding+by+the+DNA+primase+of+bacteriophage+T7.">Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7.</a> Nucleic Acids Res. 38 (13), 4372-4383 (2010).</li><li>Hernandez, A. J., Lee, S. J., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+release+is+the+rate-limiting+event+in+lagging-strand+synthesis+mediated+by+the+T7+replisome.">Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome.</a> Proc Natl Acad Sci U S A. 113 (21), 5916-5921 (2016).</li><li>Tabor, S., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+residue+in+DNA+polymerases+of+the+Escherichia+coli+DNA+polymerase+I+family+is+critical+for+distinguishing+between+deoxy-+and+dideoxyribonucleotides.">A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.</a> Proc Natl Acad Sci U S A. 92 (14), 6339-6343 (1995).</li><li>Kim, Y. T., Tabor, S., Bortner, C., Griffith, J. D., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+the+bacteriophage+T7+gene+2.5+protein.+A+single-stranded+DNA-binding+protein.">Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein.</a> J Biol Chem. 267 (21), 15022-15031 (1992).</li><li>Frick, D. N., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+primases.">DNA primases.</a> Annu Rev Biochem. 70, 39-80 (2001).</li><li>Lee, S. J., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+lysine+residues+in+the+RNA+polymerase+domain+of+the+gene+4+primase-helicase+of+bacteriophage+T7.">Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.</a> J Biol Chem. 276 (52), 49419-49426 (2001).</li><li>Cao, W., De La Cruz, 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=Quantitative+full+time+course+analysis+of+nonlinear+enzyme+cycling+kinetics.">Quantitative full time course analysis of nonlinear enzyme cycling kinetics.</a> Sci Rep. 3, 2658 (2013).</li><li>Wang, M. H., Zhao, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+determining+kinetic+constants+of+complexing+inactivation+at+identical+enzyme+and+inhibitor+concentrations.">A simple method for determining kinetic constants of complexing inactivation at identical enzyme and inhibitor concentrations.</a> FEBS Lett. 412 (3), 425-428 (1997).</li><li>Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitting+enzyme+kinetic+data+with+KinTek+Global+Kinetic+Explorer.">Fitting enzyme kinetic data with KinTek Global Kinetic Explorer.</a> Methods Enzymol. 467, 601-626 (2009).</li><li>Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+century+of+enzyme+kinetic+analysis,+1913.">A century of enzyme kinetic analysis, 1913.</a> FEBS Lett. 587 (17), 2753-2766 (2013).</li><li>Biswas, T., Resto-Roldan, E., Sawyer, S. K., Artsimovitch, I., Tsodikov, O. V. <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+non-radioactive+primase-pyrophosphatase+activity+assay+and+its+application+to+the+discovery+of+inhibitors+of+Mycobacterium+tuberculosis+primase+DnaG.">A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG.</a> Nucleic Acids Res. 41 (4), e56 (2013).</li><li>Sanyal, G., Doig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+DNA+replication+enzymes+as+targets+for+antibacterial+drug+discovery.">Bacterial DNA replication enzymes as targets for antibacterial drug discovery.</a> Expert Opin Drug Discov. 7 (4), 327-339 (2012).</li></ol>

The authors have nothing to disclose.

The most important factor in performing these experiments is the availability of highly active purified enzyme. During our work with gp4, for example, we found that storage of the purified enzyme in buffer (20 mM potassium phosphate, pH 7.4, 0.1 mM EDTA, 1 mM DTT) containing 50% glycerol at -20 &#176;C leads to a reduction in the specific activity of the preparation over a few months. Therefore we now flash-freeze small aliquots of purified gp4 in 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM TCEP, and 10% glycer...

The replication of DNA is a conserved feature of all cellular life. While the identity and number of replication components vary widely, the general mechanisms are shared across evolution1. Here, we describe gel-based, discontinuous kinetic experiments, using radioactive substrates, aimed at understanding the kinetics of lagging-strand DNA synthesis in vitro by components of the T7 replisome. The replication machinery of bacteriophage T7 is very simple, consisting of only four proteins (the DNA polymerase, gp5, and its processivity factor, E. coli thioredoxin, the bifunctional primase-helicase gp4, and the single-stranded DNA binding protein, gp2.5)2. This feature makes it an attractive model system to study the conserved biochemical mechanisms involved in DNA replication.
Special emphasis is placed on the formation of ribonucleotide primers by T7 DNA primase, a critical step in the initiation of DNA synthesis. In addition, one can also examine the usage of these primers by T7 DNA polymerase or other replication proteins by including them in the reaction mixture. The T7 primase-helicase, gp4, catalyzes the formation of primers for DNA synthesis at specific DNA sequences called primase recognition sites, or PRS, (5&#39;-GTC-3&#39;). The cytosine in the PRS is cryptic, it is essential for recognition of the site, but it is not copied into the product3. The first step in primer synthesis by gp4 involves the formation of the dinucleotide pppAC, which is then extended to a trimer, and eventually to functional tetranucleotide primers pppACCC, pppACCA, or pppACAC depending on the sequence in the template4. These primers can then be used by T7 DNA polymerase to initiate DNA synthesis, which is also a gp4-assisted process5,6. In this regard, the primase domain stabilizes the extremely short tetraribonucleotides with the template, preventing their dissociation, engages DNA polymerase in a manner conducive to securing the primer/template into the polymerase active site7. These steps (RNA primer synthesis, primer handoff to polymerase, and extension) are repeated in multiple cycles to replicate the lagging strand and must be coordinated with the replication of the leading strand.
The assays described here are highly sensitive and can be performed in a moderate time frame. However, they are relatively low-throughput and great care must be exercised in the usage and disposal of radioactive materials. Depending on the speed at which the reaction proceeds, one might employ a rapid-quench instrument to attain samples at time scales amenable for meaningful analysis of reaction time courses in either the steady or the pre-steady state. Recently, we used assays described here to provide evidence of the importance of primer release from the primase domain of gp4 in the initiation of Okazaki fragments. Additionally, we found evidence of a regulatory role for the T7 single-stranded DNA binding protein, gp2.5, in promoting efficient primer formation and utilization8.

<table border="0" cellpadding="0" cellspacing="0" width="584">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;"> 
			Tris pre-set crystals 
			pH 7.5</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">T4128&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris base</td>
			<td style="width:83px;">&#160;SIGMA</td>
			<td align="right" style="width:119px;">93362</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-Glutamic acid potassium salt monohydrate</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">G1501&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Magnesium chloride hexahydrate</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
			<td style="width:119px;">5958-04</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,4-Dithiothreitol</td>
			<td style="width:83px;">J.T. Baker</td>
			<td style="width:119px;">F780-02</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sodium Dodecyl Sulfate</td>
			<td style="width:83px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">811030</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">(Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
			<td style="width:119px;">4931-04</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">[&#945;-32P] CTP</td>
			<td style="width:83px;">PerkinElmer</td>
			<td style="width:119px;">BLU508H</td>
			<td>radioactive, take protective measures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">[&#947;-32P] ATP</td>
			<td style="width:83px;">PerkinElmer</td>
			<td style="width:119px;">BLU502A</td>
			<td>radioactive, take protective measures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 mM dATP, dCTP, dGTP, dTTP&#160;</td>
			<td style="width:83px;">Affymetrix</td>
			<td align="right" style="width:119px;">77100</td>
			<td>four dNTPs part of set</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 mM ATP and CTP</td>
			<td style="width:83px;">Epicentre</td>
			<td style="width:119px;">RN02825</td>
			<td>part of NTP set</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">ssDNA constructs</td>
			<td style="width:83px;">Integrated DNA Technologies</td>
			<td style="width:119px;">N/A</td>
			<td>custom sequences&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">methanol&#160;</td>
			<td style="width:83px;">Macron Fine Chemicals</td>
			<td style="width:119px;">3017-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">formamide</td>
			<td style="width:83px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">17899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bromophenol blue</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">B8026&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cylene xyanol&#160;</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">X4126&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acrylamide</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">A9099</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N,N&#8242;-Methylenebisacrylamide</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">M7279&#160;</td>
			<td>Toxic</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Urea</td>
			<td style="width:83px;">Boston Bioproducts</td>
			<td style="width:119px;">P-940</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Boric acid</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
			<td align="right" style="width:119px;">2549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rapid Quench-Flow Instrument&#160;</td>
			<td style="width:83px;">Kintek Corp.</td>
			<td style="width:119px;">RQF-3&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kintek Explorer</td>
			<td style="width:83px;">Kintek Corp.</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kaleidagraph&#160;</td>
			<td style="width:83px;">Synergy Software</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

kinetics of lagging-strand dna synthesis in vitro by the bacteriophage t7 replication proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

The results shown in Figure 1A were obtained as described in step 1 of the protocol, i.e., by manually sampling a primer synthesis reaction catalyzed by gp4 under multiple-turnover conditions. Here, a range of products after gel electrophoresis can be observed by using CTP labeled with 32P at the &#945;-position in primer synthesis reactions (Figure 1A). The labeled precursor, CTP, shows the highest mobility and migrat...

Watch this Scientific Journal Video about Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins at JoVE.com

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

kinetics-lagging-strand-dna-synthesis-vitro-bacteriophage-t7

Research

Education

JoVE Core

JoVE Journal

Molecular Biology

Genetics

Unit 1

DNA Replication

SCIENTISTS IN ACTION

7.3K Views.  Harvard Medical School. We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7. 

Video: Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...