We thank Dr T. Helleday and Dr E. Petermann for help with the fiber technique as well as members of the lab for helpful discussion. WN is supported by a Senior International Research Fellowship from the Association for International Cancer Research and by the Polish State Committee for Scientific Research grant (N301 165935).

The method described here was used in the following publication: Schwab et al., EMBO J., 29(4):806-185.
1. DT40 cell culture
Materials: Fetal bovine serum (FBS), chicken serum, penicillin/streptomycin, 2-mercaptoethanol, RPMI
<ol>
<li>Prepare cell culture medium: add 7% FBS, 3% chicken serum, 1x penicillin/streptomycin and 10 μM 2-mercaptoethanol to RPMI medium.</li>
<li>DT40 cells are grown in sterile cell culture flasks at 38 °C and 5% CO2 in a humidified incubator. Split cells every day to a density of 5 x 105 cells/ml6.</li>
</ol>
2. In vivo labeling
Materials: IdU, CldU
<ol>
<li>Prepare stock solutions of nucleotide analogues: dissolve IdU to 5 mM and CldU to 2.5 mM in medium. Heat both solutions briefly to 60 °C and vortex until the nucleotide analogues are dissolved completely.</li>
<li>Add IdU to a final concentration of 25 μM to exponentially growing DT40 cells and mix the cell suspension well. Incubate cells for 20 minutes at 38 °C and 5% CO2.</li>
<li>After incubation with the first label, add CldU to a final concentration of 250 μM and treat cells as described in the previous step.</li>
<li>Wash cells with ice cold PBS and resuspend them at a concentration of 7.5 x 105 cells/ml in cold PBS. Keep the labeled cells on ice.</li>
</ol>
The labeling procedure described is merely a suggestion and can be modified to address specific questions. Please refer to the discussion section for more information on experimental design.
3. Cell lysis and DNA spreading
Materials: Glass slides, fiber lysis solution
<ol>
<li>Prepare the fiber lysis solution: 50 mM EDTA and 0.5% SDS in 200 mM Tris-HCl, pH 7.5</li>
<li>Spot 2 μl of the cell suspension to one end of the glass slide. Air-dry for 5 minutes or until the volume of the drop is greatly reduced but not dry.</li>
<li>Pipet 7 μl lysis solution on top of the cell suspension and gently stir with the pipette tip to mix the solutions. Incubate for 2 minutes for cell lysis to proceed.</li>
<li>Tilt slides to 15° to allow the fibers to spread along the slide.</li>
<li>Once the fiber solution has reached the bottom of the slide, place it horizontally to air-dry. After drying, a thin, opaque line should be visible along the slide. At this point, the beginning of the stretched fibers should be marked with a pencil as after staining the line will not be visible any longer. This mark will later help to locate the fibers under the microscope.</li>
</ol>
4. Immunofluorescence staining
Materials: Methanol, acetic acid, HCl, 5% BSA in PBS, staining jar, anti-BrdU (mouse) antibody, anti-BrdU (rat) antibody, sheep anti-mouse Cy3 antibody, goat anti-rat Alexa Fluor 488 antibody, Vectashield mounting medium, coverslips, nail polish
<ol>
<li>Immerse slides in methanol/acetic acid (3:1) in a staining jar and incubate for 10 minutes.</li>
<li>Wash slides in distilled H2O, then immerse in 2.5 M HCl for 80 minutes.</li>
<li>Wash slides three times in PBS for 5 minutes.</li>
<li>Remove slides from the staining jar and collect excess PBS with a paper towel. Place the slides horizontally and pipet 5% BSA on top of each slide. Cover the slides gently with a coverslip to spread the BSA evenly over the slide and incubate for 20 minutes.</li>
<li>Dilute the primary antibodies in 5% BSA at the following concentrations: 1:25 anti-BrdU (mouse) and 1:400 anti-BrdU (rat).</li>
<li>Move the coverslip gently down the glass slide to remove it. Do not apply force if the cover slip sticks to the slide. The slide can be rehydrated in PBS until the coverslip becomes loose and can be removed with ease. Collect excess BSA with a paper towel and place the slides horizontally. Pipet 50 μl of the primary antibody solution onto each slide. Cover again with a coverslip to spread the antibody solution evenly over the slide and incubate in a humidified chamber for 2 hours.</li>
<li>After removing the coverslips, wash the slides three times in PBS for 5 minutes</li>
<li>Dilute the secondary antibodies in 5% BSA at the following concentrations: 1:500 sheep anti-mouse Cy3 and 1:400 goat anti-rat Alexa Fluor 488.</li>
<li>Apply 50 μl of the secondary antibody solution as described for the primary antibodies. Protect the slides from light and incubate for 1 hour.</li>
<li>After removing the coverslips, wash the slides three times in PBS for 5 minutes.</li>
<li>Add a drop of Vectashield mounting medium onto each slide and apply a coverslip. Gently press the coverslips and remove excess fluid around it with a paper towel. Seal coverslips with transparent nail polish and let them dry. Store the slides at -20 °C.</li>
</ol>
5. Image acquisition
Materials: fluorescence microscope, camera
Place a drop of immersion oil onto a slide close to the pencil mark and start locating the fibers. Typically, there is a main fiber bundle, but these fibers are too entangled and cannot be analyzed later. Move away from the main bundle to find areas where fibers are clearly separated from each other (Fig. 2). We would select pictures only using one color channel in order to avoid bias. Then, we would take approximately 10 pictures of each time point, concentration or cell line. However, the number of pictures depends on how many fibers can be counted on each picture. Move along the slide to take the different pictures, as one area of a slide may not provide representative fiber lengths or replication structures.
6. Data analysis
Materials: Picture analysis program, e.g. ImageJ (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) 
Import pictures into a picture analysis program. Measure the lengths of the fiber tracts and/or count the different replication structures (Fig 3). Only count fibers which are clearly visible and which do not extend over the edge of the picture. We typically measure the length of about 100 fibers and count 150-200 replication structures and we would repeat individual experiments at least three times.
7. Representative Results: 
Newly replicated DNA can be visualized as lines of antibody-labeled nucleotide analogues. In our experiments an ongoing fork is represented as adjacent red and green signals (Fig 3). The double labeling protocol also allows us to define four major classes of replication structures: 1) new initiation events can be divided into origins that have fired while the cells were incubated with the first label and origins that have fired during the incubation with the second label. The former consist of neighboring green-red-green signals and the latter of a green line only. 2) termination events manifest as adjoining red-green-red signals. 3) interspersed origins are sites in the genome with closely spaced origins. Such sites consist of consecutive origins and termination signals. 4) depending on the experimental design, stalled/collapsed forks can either be defined as a red only signal7 or a red line followed by a short green tract8,9.
Using the conditions described here, wild type DT40 cells have an average fork speed of 0.4 μm/min. We can detect approximately 63% ongoing forks, 10% origins, 16% stalled forks (red only tracts), 8% terminations and 3% interspersed fibers. 
<img src="/files/ftp_upload/3255/3255fig1.jpg" alt="DNA replication with IdU/CldU, diagram shows experimental labeling and fluorescence analysis." data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. (A) The left hand side of the cartoon depicts the first step of the DNA labeling procedure: adding IdU to exponentially growing DT40 cells leads the nucleotide analogue to be readily incorporated into the newly synthesized leading and lagging DNA strands. This can be visualized by using a specific antibody, and will appear as a red line when viewed under the microscope. Subsequently, the same procedure is repeated with CldU as shown on the right side of the figure. After incubation with the second nucleotide analogue, an active fork will be visible as an adjoining red-green tract. The average fiber length is proportional to the incubation time of the nucleotide analogues. (B) DNA from lysed DT40 cells is stretched by gravity onto a microscope slide and the incorporated nucleotide analogues are visualized by use of specific antibodies and fluorescence microscopy. 
<img src="/files/ftp_upload/3255/3255fig2.jpg" alt="Fluorescence microscopy image; cytoskeleton fibers in red and green; cellular structure analysis." data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. A representative fluorescence image shows fibers from wild type DT40 cells subsequently labeled with IdU and CldU for 20 minutes each. Most of the fibers are well separated from each other and can thus be easily analyzed. The white bar represents 10 μm. 
<img src="/files/ftp_upload/3255/3255fig3.jpg" alt="DNA replication forks diagram showing origin, termination, and stalled fork processes." data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. The double-labeling fiber technique allows one to distinguish between different replication structures; 1 - actively replicating forks, 2 - new sites of DNA replication (firing of new origins), 3 - fork terminations (two converging forks), 4 - interspersed fibers (sites of closely spaced origins) and 5 - stalled forks (red only signal).

<ol>
	<li>Caldwell, R. B., Fiedler, P., Schoetz, U., Buerstedde, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+function+analysis+using+the+chicken+B-cell+line+DT40.">Gene function analysis using the chicken B-cell line DT40.</a> Methods. Mol. Biol. 408, 193-210 (2007).</li><li>Petes, T. D., Williamson, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+autoradiography+of+replicating+yeast+DNA.">Fiber autoradiography of replicating yeast DNA.</a> Exp. Cell. Res. 95, 103-110 (1975).</li><li>Takeuchi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+frequency+of+initiation+sites+of+DNA+replication+in+Werner's+syndrome+cells.">Altered frequency of initiation sites of DNA replication in Werner's syndrome cells.</a> Hum. Genet. 60, 365-368 (1982).</li><li>Jackson, D. A., Pombo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicon+clusters+are+stable+units+of+chromosome+structure:+evidence+that+nuclear+organization+contributes+to+the+efficient+activation+and+propagation+of+S+phase+in+human+cells.">Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells.</a> J. Cell. Biol. 140, 1285-1295 (1998).</li><li>Schwab, R. A., Blackford, A. N., Niedzwiedz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR+activation+and+replication+fork+restart+are+defective+in+FANCM-deficient+cells.">ATR activation and replication fork restart are defective in FANCM-deficient cells.</a> EMBO. J. 29, 806-818 (2010).</li><li>Saribasak, H., Arakawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+cell+culture+conditions.">Basic cell culture conditions.</a> Subcell. Biochem. 40, 345-346 (2006).</li><li>Conti, C., Seiler, J. A., Pommier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+DNA+replication+elongation+checkpoint:+implication+of+Chk1+and+relationship+with+origin+firing+as+determined+by+single+DNA+molecule+and+single+cell+analyses.">The mammalian DNA replication elongation checkpoint: implication of Chk1 and relationship with origin firing as determined by single DNA molecule and single cell analyses.</a> Cell. Cycle. 6, 2760-2767 (2007).</li><li>Petermann, E., Orta, M. L., Issaeva, N., Schultz, N., Helleday, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyurea-stalled+replication+forks+become+progressively+inactivated+and+require+two+different+RAD51-mediated+pathways+for+restart+and+repair.">Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.</a> Mol. Cell. 37, 492-502 (2010).</li><li>Edmunds, C. E., Simpson, L. J., Sale, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCNA+ubiquitination+and+REV1+define+temporally+distinct+mechanisms+for+controlling+translesion+synthesis+in+the+avian+cell+line+DT40.">PCNA ubiquitination and REV1 define temporally distinct mechanisms for controlling translesion synthesis in the avian cell line DT40.</a> Mol. Cell. 30, 519-529 (2008).</li></ol>

No conflicts of interest declared.

We describe a method that allows for quantitative analysis of parameters that influence DNA synthesis during S-phase at a single molecule level. Over the past ten years, different versions of the DNA fiber fluorography technique were developed to visualize the movement of individual replication forks within living cells. The critical parameter in all these techniques is the procedure used to achieve the best possible stretching of DNA on glass surfaces providing well-separated DNA fibers. A crucial step to achieve this is the incubation time of the cell suspension on the microscope slide as well as lysis time. These parameters depend on the temperature and airflow in a particular lab and should be determined empirically. The practical part of a DNA fiber experiment is typically accomplished within one day. However, subsequent picture acquisition and analysis is more time consuming and requires practice.
The labeling protocol can be adapted to answer various scientific problems: using an agent that interferes with replication during the second pulse labeling monitors fork elongation/stalling in response to replicative stress. In this scenario, the first label does not need to be washed out as the second nucleotide is added in excess. Alternatively, drugs that impede replication can be used in combination or just after addition of the first label. This requires the first label and the drug to be removed before the second nucleotide analogue is applied. This procedure allows one to determine the importance of various DNA repair factors on replicative fork stability/recovery under conditions of replicative stress (i.e fork stalling and/or collapse). Noteworthy, a more detailed analysis of particulars of the DNA replication program could be performed with the use of an alternative approaches combining DNA combing and the fluorescence in situ hybridization. This however, is technically more challenging and requires expensive equipment. 
The ability to qualitatively and quantitatively analyze particulars of DNA replication provides an essential tool for investigating defects in DNA replication itself as well as the pathways that suppress genomic instability associated with replication forks. Undoubtedly, this assay will further help to shed light on the mechanisms of replication-mediated DNA repair that allow normal cells to respond/adapt to environmental changes as well as how cancer cells utilize these mechanisms to resist the toxicity of drugs targeting replication forks.

<table><tbody><tr><td>Fetal bovine serum gold (FBS)</td><td>PAA Laboratories</td><td>A15-151</td><td></td></tr><tr><td>Chicken serum</td><td>Sigma-Aldrich</td><td>C5405</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>PAA Laboratories</td><td>P11-010</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO, by Life Technologies</td><td>21875</td><td></td></tr><tr><td>5-Iodo-2&amp;rsquo;-deoxyuridine (IdU)</td><td>Sigma-Aldrich</td><td>I7125</td><td></td></tr><tr><td>5-Chloro-2&amp;rsquo;-deoxy-uridine (CldU)</td><td>Sigma-Aldrich</td><td>C6891</td><td></td></tr><tr><td>Microscope slides SuperFrost</td><td>VWR international</td><td>631-0910</td><td></td></tr><tr><td>Cover slips No1</td><td>Scientific Laboratory Supplies LTD</td><td>MIC3234</td><td></td></tr><tr><td>Vectashield mounting medium</td><td>H-1000 Vector lab</td><td>H-1000</td><td></td></tr><tr><td>Anti-BrdU antibody (mouse)</td><td>BD Biosciences</td><td>347580</td><td></td></tr><tr><td>Anti-BrdU antibody (rat)</td><td>Abcam</td><td>ab6326</td><td></td></tr><tr><td>sheep anti-mouse Cy3</td><td>Sigma-Aldrich</td><td>C2181</td><td></td></tr><tr><td>goat anti-rat Alexa Fluor 488 antibody</td><td>Invitrogen</td><td>A110060</td><td></td></tr></tbody></table>

visualization of dna replication in the vertebrate model system dt40 using the dna fiber technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

Watch this Scientific Journal Video about Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique at JoVE.com

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

visualization-dna-replication-vertebrate-model-system-dt40-using-dna

Research

JoVE Journal

Biology

39.8K Views.  University of Oxford. DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Video: Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

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

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

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

Genetics

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

2D Gel Electrophoresis for Analyzing DNA Replication and Fork Stalling Events

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA. Intermediates are then digested by appropriate restriction enzymes to place the repeat of interest within the origin-distal half of a 3-5 kb-long DNA fragment. The replication intermediates are separated into two perpendicular dimensions, first by size and then by shape. Following Southern blot hybridization, this approach allows researchers to observe fork stalling at various structure-forming repeats on the descending half of the replication Y-arc. Furthermore, this positioning of the stall site allows the visualization of various outcomes of repeat-mediated fork stalling, such as fork reversal, the advent of a converging fork, and recombinational fork restart.

Author Spotlight: Characterizing DNA Replication of Pathogenic Repeats to Uncover Mechanisms of Replication Fork Stalling and Expansion

Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This ...

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

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

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

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

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

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

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

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

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

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

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

Biochemistry

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles

Nanomaterial exposure can cause replication stress and genomic instability in cells. The degree of instability depends on the chemistry, size, and concentration of the nanomaterials, the time of exposure, and the exposed cell type. Several established methods have been used to elucidate how endogenous/exogenous agents impact global replication. However, replicon-level assays, such as the DNA fiber assay, are imperative to understand how these agents influence replication initiation, terminations, and replication fork progression. Knowing this allows one to understand better how nanomaterials increase the chances of mutation fixation and genomic instability. We used RAW 264.7 macrophages as model cells to study the replication dynamics under graphene oxide nanoparticle exposure. Here, we demonstrate the basic protocol for the DNA fiber assay, which includes pulse labeling with nucleotide analogs, cell lysis, spreading the pulse-labeled DNA fibers onto slides, fluorescent immunostaining of the nucleotide analogs within the DNA fibers, imaging of the replication intermediates within the DNA fibers using confocal microscopy, and replication intermediate analysis utilizing a computer-assisted scoring and analysis (CASA) software.

The methodology assists in the analysis of variation in DNA replication dynamics in the presence of nanoparticles. Different methodologies can be adopted based on the cytotoxicity level of the material of interest. In addition, a description of the image analysis is provided to help in DNA fiber analysis.

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Summary

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Chapters in this video

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome

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

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

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

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles