Name: Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins (Video) | JoVE Journal
Uploaded: 2017-02-25T15:00:00.000+00:00
Duration: 8 min 14 s
Description: Learn gel-based assays to examine kinetics of lagging-strand DNA synthesis using bacteriophage T7 replication proteins. This method identifies key steps during Okazaki fragment synthesis.

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5 minutes

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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKornberg%2C+A+%22DNA+replication%22">DNA replication</a>. , 2nd, W.H. Freeman. (1992).</li>
<li>Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motors%2C+switches%2C+and+contacts+in+the+replisome%5BTitle%5D)+AND+%22Annu+Rev+Biochem%22%5BJournal%5D)">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/pubmed?term=((Roles+of+bacteriophage+T7+gene+4+proteins+in+providing+primase+and+helicase+functions+in+vivo%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Primer+initiation+and+extension+by+T7+DNA+primase%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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/pubmed?term=((Modular+architecture+of+the+bacteriophage+T7+primase+couples+RNA+primer+synthesis+to+DNA+synthesis%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">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/pubmed?term=((Gene+4+DNA+primase+of+bacteriophage+T7+mediates+the+annealing+and+extension+of+ribo-oligonucleotides+at+primase+recognition+sites%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((Mechanism+of+sequence-specific+template+binding+by+the+DNA+primase+of+bacteriophage+T7%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">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/pubmed?term=((Primer+release+is+the+rate-limiting+event+in+lagging-strand+synthesis+mediated+by+the+T7+replisome%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((A+single+residue+in+DNA+polymerases+of+the+Escherichia+coli+DNA+polymerase+I+family+is+critical+for+distinguishing+between+deoxy-+and+dideoxyribonucleotides%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Purification+and+characterization+of+the+bacteriophage+T7+gene+2.5+protein.+A+single-stranded+DNA-binding+protein%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((DNA+primases%5BTitle%5D)+AND+%22Annu+Rev+Biochem%22%5BJournal%5D)">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/pubmed?term=((Essential+lysine+residues+in+the+RNA+polymerase+domain+of+the+gene+4+primase-helicase+of+bacteriophage+T7%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((Quantitative+full+time+course+analysis+of+nonlinear+enzyme+cycling+kinetics%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">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/pubmed?term=((A+simple+method+for+determining+kinetic+constants+of+complexing+inactivation+at+identical+enzyme+and+inhibitor+concentrations%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">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/pubmed?term=((Fitting+enzyme+kinetic+data+with+KinTek+Global+Kinetic+Explorer%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">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/pubmed?term=((A+century+of+enzyme+kinetic+analysis%2C+1913%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">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/pubmed?term=((A+novel+non-radioactive+primase-pyrophosphatase+activity+assay+and+its+application+to+the+discovery+of+inhibitors+of+Mycobacterium+tuberculosis+primase+DnaG%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">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/pubmed?term=((Bacterial+DNA+replication+enzymes+as+targets+for+antibacterial+drug+discovery%5BTitle%5D)+AND+%22Expert+Opin+Drug+Discov%22%5BJournal%5D)">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><tbody><tr><td>Tris pre-set crystals&lt;br/&gt; pH 7.5</td><td>SIGMA</td><td>T4128</td><td></td></tr><tr><td>Tris base</td><td>SIGMA</td><td>93362</td><td></td></tr><tr><td>L-Glutamic acid potassium salt monohydrate</td><td>SIGMA</td><td>G1501&amp;nbsp;</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Mallinckrodt Chemicals</td><td>5958-04</td><td></td></tr><tr><td>1,4-Dithiothreitol</td><td>J.T. Baker</td><td>F780-02</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>MP Biomedicals</td><td>811030</td><td></td></tr><tr><td>(Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate</td><td>Mallinckrodt Chemicals</td><td>4931-04</td><td></td></tr><tr><td>[&amp;alpha;-&lt;sup&gt;32&lt;/sup&gt;P] CTP</td><td>PerkinElmer</td><td>BLU508H</td><td>radioactive, take protective measures</td></tr><tr><td>[&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P] ATP</td><td>PerkinElmer</td><td>BLU502A</td><td>radioactive, take protective measures</td></tr><tr><td>100 mM dATP, dCTP, dGTP, dTTP&amp;nbsp;</td><td>Affymetrix</td><td>77100</td><td>four dNTPs part of set</td></tr><tr><td>100 mM ATP and CTP</td><td>Epicentre</td><td>RN02825</td><td>part of NTP set</td></tr><tr><td>ssDNA constructs</td><td>Integrated DNA Technologies</td><td>N/A</td><td>custom sequences&amp;nbsp;</td></tr><tr><td>methanol&amp;nbsp;</td><td>Macron Fine Chemicals</td><td>3017-08</td><td></td></tr><tr><td>formamide</td><td>Thermo Scientific</td><td>17899</td><td></td></tr><tr><td>bromophenol blue</td><td>SIGMA</td><td>B8026&amp;nbsp;</td><td></td></tr><tr><td>cylene xyanol&amp;nbsp;</td><td>SIGMA</td><td>X4126&amp;nbsp;</td><td></td></tr><tr><td>acrylamide</td><td>SIGMA</td><td>A9099</td><td>Toxic</td></tr><tr><td>&lt;em&gt;N,N&amp;prime;&lt;/em&gt;-Methylenebisacrylamide</td><td>SIGMA</td><td>M7279&amp;nbsp;</td><td>Toxic</td></tr><tr><td>Urea</td><td>Boston Bioproducts</td><td>P-940</td><td></td></tr><tr><td>Boric acid</td><td>Mallinckrodt Chemicals</td><td>2549</td><td></td></tr><tr><td>Rapid Quench-Flow Instrument&amp;nbsp;</td><td>Kintek Corp.</td><td>RQF-3&amp;nbsp;</td><td></td></tr><tr><td>Kintek Explorer</td><td>Kintek Corp.</td><td>N/A</td><td></td></tr><tr><td>Kaleidagraph&amp;nbsp;</td><td>Synergy Software</td><td>N/A</td><td></td></tr></tbody></table>

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

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 α-position in primer synthesis reactions (Figure 1A). The labeled precursor, CTP, shows the highest mobility and migrates to...

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

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

The overall goal of this procedure is to examine the kinetics of primer and lagging strand DNA synthesis. This method can help answer key questions in the DNA replication field, such as how primers form by DNA primase, are transferred to, and utilized by, DNA polymerase. The main advantage of this technique is that it allows the identification of key steps during Okazaki fragment synthesis.Since radioactive materials are used in this experiment, all institutional regulations regarding the safe use and disposal of radioactive materials must be followed. Start by aliquoting two microliters of formamide dye buffer into eight 1.5 milliliter polypropylene tubes, equal to the number of time points to be analyzed. Next, prepare a 20 microliter mixture consisting of buffer, ATP, CTP, CTP labeled on the alpha-phosphate group by P32, single-strand DNA template, and the gene four protein, or GP four primase helicase.Incubate the mixture at room temperature for five minutes. After five minutes, use a pipettor to remove two microliters of the mixture, and mix it with two microliters of formamide dye buffer in one of the 1.5 milliliters tubes prepared earlier. This is the no reaction control.Add two microliters of 0.1 molar magnesium chloride to the reaction mixture and immediately start a timer. At 10 second intervals, manually withdraw two microliter samples of the reaction mixture, and mix with the formamide dye in the 1.5 milliliter tubes. After all time points have been collected, heat the samples in a heat block at 95 degrees Celsius for five minutes.Load aliquots of the samples onto a denaturing gel for electrophoresis. Begin this procedure by preparing the reaction mixtures. Prepare 400 microliters of solution A containing buffer, GP four hexamer, single-strand DNA template, dNTPs, ATP, CTP, and CTP labeled on the alpha-phosphate group by P32.Prepare 400 microliters of solution B, containing one X buffer and 20 millimolar of magnesium chloride. Next, set up the rapid quench flow instrument. Turn on the instrument, and after the main menu appears, type seven on the instrument keyboard to move the syringe driver to the home position.Open the buffer syringe position to load. Fill syringe C with quench solution, then, using a 10 milliliter syringe, fill the instrument buffer reservoirs A and B with buffer. Remove air bubbles by pushing plungers in and out.Change the knob positions to fire. Type two to adjust the position of the syringe driver bar. Press the adjust down button to lower the syringe until the buffer comes out the exit loop.Type escape to return to the main menu. To wash the tubing, attach the exit line to a vacuum. Change the sample knobs to flush.Set the knobs to the horizontal position to close the buffer syringe. Turn on the vacuum, dip the two flush loops in water for 10 seconds, and then dip in methanol for 10 seconds. Dry the loops by sucking air for about 15 seconds, or until dry.To load samples, change the sample knob position to load. Place solution A into a one milliliter syringe, plug the syringe into one of the sample load ports, in the same way, place solution B in the opposite sample load port. Begin the collection of time points by typing one on the main menu for a quench flow run.Enter the reaction time and press enter. The reaction loop number to use will be displayed. Switch to the required reaction loop.After washing the tube as demonstrated earlier, turn the sample knobs to load. Load the reaction mixes in the sample loops until just outside the central mixing compartment. Turn all the knobs to fire, and confirm that all knobs are in the correct position.Place a 1.5 milliliter tube onto the end of the exit loop. Press go to run the reaction. After repeating the tubing wash procedure, reload buffer syringes A and B until they hit the buffer syringe driver.Repeat the steps for running the reaction for each desired time point, with one exception, do not load solution B when quenching the zero time point control. When the sample collection is finished, enter escape to return to the main menu. Press seven to return the buffer syringe driver to its home position.Start the instrument clean up by removing the reaction mixture syringes, turn the sample loading knob to the load position. Under vacuum, wash each sample loop with 10 milliliters each of sodium hydroxide, phosphoric acid, and methanol. Press down on all three syringe plungers to retrieve the reaction and quench buffers.Wash the syringes with five milliliters of water at least twice. Fill the syringes again with five milliliters of water, turn the knobs to the fire position, turn on the vacuum and bring down the driver via the keyboard command to remove the water. Wash the tubing as demonstrated earlier.Lastly, turn off the rapid quench flow instrument. The samples are subsequently analyzed as described in the text protocol. These results were obtained by manually sampling a primer synthesis reaction catalyzed by GP four under multiple turnover conditions.The labeled precursor CTP shows the highest mobility and migrates towards the bottom of the gel, followed by slower migrating species, corresponding to the dye and tetraribonucleotides synthesized by GP four. Depending on the length of incubation and template sequence context, additional labeled products corresponding to higher oligomers can be observed. When a rapid quench instrument is used to examine the reaction catalyzed by GP four in timescales in the range of seconds down to a few ms, it is evident that primer accumulation is not linear, but displays a biphasic profile indicated by the solid line.This suggests that product formation is rapid, and release is rate-limiting in the steady state. The dotted line represents a hypothetical progress curve that would result if primer formation by GP four did not proceed with a pre-steady state burst. A single-turnover primer synthesis experiment provides information about the formation and decay of intermediate species during the conversion of substrate to product.A time course of tetramer formation is shown by the gel image. The graph shows intermediate formation versus time. Once mastered, this technique can be done in 90 minutes, if it's performed properly.Don't forget that working with radioactive reagents can be extremely hazardous and precautions such as proper shielding should always be taken while performing this procedure.

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Genetics

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Learn gel-based assays to examine kinetics of lagging-strand DNA synthesis using bacteriophage T7 replication proteins. This method identifies key steps during Okazaki fragment synthesis.

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