Name: kinetics of lagging-strand dna synthesis in vitro by the bacteriophage t7 replication proteins
Uploaded: 2017-02-25T15:00:00
Description: Watch this Scientific Journal Video about Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins at JoVE.com

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 described<sup class="xre...

<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 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.

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			Tris pre-set crystals 
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			<td style="width:83px;">Mallinckrodt Chemicals</td>
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			<td height="21" style="height:21px;width:216px;">N,N&#8242;-Methylenebisacrylamide</td>
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			<td height="63" style="height:63px;width:216px;">Boric acid</td>
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			<td height="42" style="height:42px;width:216px;">Rapid Quench-Flow Instrument&#160;</td>
			<td style="width:83px;">Kintek Corp.</td>
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			<td height="42" style="height:42px;width:216px;">Kintek Explorer</td>
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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 ...

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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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 ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

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 ...