Name: genome-wide determination of mammalian replication timing by dna content measurement
Uploaded: 2017-01-19T14:00:00.000+00:00
Duration: 8 min 6 s
Description: Watch this Scientific Journal Video about Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement at JoVE.com

We thank Oriya Vardi for assistance in generating figures. Work in the IS group was supported by the Israel Science Foundation (grant No. 567/10) and the European Research Council Starting Grant (#281306).

Note: ToR can be measured only on growing, unsynchronized cells. The procedure should begin with at least 1 - 2 x 106 fast growing cells, which will usually result in ~1 x 105 cells in S phase (the rate limiting step). It is recommended to conduct each experiment using two or three replicates. The entire process of CNR-ToR can be completed within one week &#8212; two days should be dedicated to all steps up to library preparation, one to two days are needed for sequencing and an additional day is necessary for the initial data analysis.
1. Collection of Cells from Culture
NOTE: The protocol is written for cells growing in culture in 10 cm plates (containing approximately 2 - 5 x 106 cells), but can be easily adjusted to other platforms.
<ol>
	<li>For cells that were grown in suspension, proceed to fixation (section 2).</li>
	<li>For adherent cells, aspirate and wash the plate with 3 mL PBS without Ca2+ and Mg2+.</li>
	<li>Discard the PBS and incubate the cells for 5 min at 37 &#176;C with 1 mL commercial trypsin-EDTA until cells detach. 
	NOTE: The duration of the trypsin treatment should be adjusted to each cell type.</li>
	<li>Add 3 mL culture media to neutralize the trypsin and collect cells in a 15 mL conical tube or a 5 mL polystyrene tube. Keep on ice.</li>
</ol>
2. Fixation
NOTE: For this part, all steps should be done at 4 &#176;C.
<ol>
	<li>Centrifuge the cells at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate and wash cells twice with 1 mL cold PBS.</li>
	<li>Resuspend cells in 250 &#181;L (total) cold PBS.</li>
	<li>While gently vortexing the tube, slowly add dropwise 800 &#181;L of -20 &#176;C 100% ethanol. This leads to a final ethanol concentration of 70 - 80%. 
	NOTE: High purity ethanol is recommended at this step.</li>
	<li>Incubate cells on ice for 30 min. 
	NOTE: At this stage cells can be kept for a few days at 4 &#176;C or for a few months at -20 &#176;C.</li>
</ol>
3. PI Staining
<ol>
	<li>Centrifuge the cells at 500 x g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant carefully and wash cells twice with 1 mL cold PBS.</li>
	<li>Aspirate and resuspend each sample with the following mixture: 1 mL PBS, 5 &#181;L 10 mg/mL RNaseA, 50 &#181;L 1 mg/mL propidium iodide (PI; mix bottle before use). Adjust concentration up to ~2 x 106 cells/mL). 
	NOTE: Keep out of light &#8212; PI is light sensitive.</li>
	<li>Filter through 35 &#181;m mesh to a 5 mL polystyrene tube, and close with Parafilm.</li>
	<li>Incubate at RT in the dark, for 15 - 30 min. 
	NOTE: Cells are now ready for FACS analysis. If necessary, the stained cells can be stored for at least 24 h at 4 &#176;C in the dark.</li>
</ol>
4. Sort
<ol>
	<li>Sort cells using a FACS machine. Use the 561 nm laser to differentiate cells based on their PI intensity. Other lasers near the 535 nm excitation maximum like 488 nm or 532 can be used depending on the FACS machine configuration.</li>
	<li>For optimal results, use the smallest nozzle recommended for the specific cell size (for most cells 85 &#181;m). Prioritize purity modes over yield. Use a steady slow flow, usually up to 300-500 events/s, with a sheath pressure of 45 psi.</li>
	<li>Using gating, discriminate dead cells and subcellular debris (low FCS and high SSC) by Plotting FCS vs SSC. From the viable cells discriminate doublets by plotting SSC-Width (W) vs SSC-Height (H) followed by a FSC-W vs FSC-H plot and by PI- W vs PI-H (doublets will have the same H-value but bigger W-value). For the viable single cells draw a histogram of the PI-Area (A) intensity which represents the DNA content of the cells.</li>
	<li>Sort cells into G1 and S phases, as shown in Figure 1. Gating for S should be wide and intrude into the G1 and G2 phases, while G1 gating should be narrow and as far from S as possible.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55157/55157fig1.jpg" /> 
Figure 1. Cell cycle phase determination based on PI intensity. Histogram showing the distribution of the cellular DNA content (measured by PI-Area) of mouse embryonic fibroblast (MEF) population. The DNA content is used to sort the population into two sub-populations i) G1 cells (2N DNA content) and ii) S phase cells (2N - 4N DNA content), using the marked regions. <a href="http://cloudfront.jove.com/files/ftp_upload/55157/55157fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: The purpose of the collection of G1 cells is to account for biases in the sequencing efficiency between different genomic regions. An alternative approach is to use G1 arrested cells from the same cell type. This approach gives cleaner results (since it minimizes S phase contamination) but it may introduce biases stemming from genetic differences between the arrested cells and the measured cells.
<ol start="5">
	<li>Sort in cold conditions and collect sorted cells into 1.5/2 mL tubes. Keep tubes on ice following the sort. 
	NOTE: In order to improve DNA recovery, it is better to use low binding tubes or to use tubes coated with 4 - 5% BSA for 1 - 3 h at 4 &#176;C 18.</li>
</ol>
5. DNA Purification
<ol>
	<li>For each sample (G1 and S) purify DNA using a DNA purification kit. 
	NOTE: When using the commercial kit, elute with 400 &#181;L elution buffer into a 2 mL tube for high yield, as recommended by the manufacturer.</li>
	<li>Check DNA concentration using fluorometer. 
	NOTE: From 100,000 mammalian cells collected by the FACS, one should get ~ 1 &#181;g of DNA. At this stage DNA can be kept for a few days at 4 &#176;C or at -20 &#176;C for long storage.</li>
</ol>
6. Sonication
<ol>
	<li>Transfer DNA into a 1.7 mL tube that is compatible with the magnetic stand used.</li>
	<li>Concentrate DNA using 2x SPRI beads according to the manufacturer&#39;s instructions, using a magnetic stand, and elute in 50 &#181;L elution buffer.</li>
	<li>Shear DNA with a focused ultrasonicator to an average target peak size of 250 bp. Use the following settings for a 50 &#181;L DNA sample: 50 W, 20% Duty factor, 200 Cycles per burst, 20 &#176;C, 120 s.</li>
	<li>Verify the size of the sheared DNA by electrophoresis. The recommended size distribution is 200 - 700 bp with a peak at ~ 250 bp. 
	NOTE: At this stage DNA can be kept for a few days at 4 &#176;C or at -20 &#176;C for long storage.</li>
</ol>
7. Library Preparation, and Sequencing
NOTE: Many library preparation kits and different sequencing platforms should work similarly to the ones used by us and mentioned in the materials section. Actually in the past, ToR maps were generated using a very similar method with microarray platforms2.
<ol>
	<li>Prepare libraries using any commercial library preparation kit.</li>
	<li>At the end of library preparation, select size using magnetic beads for 300 - 800 bp.</li>
	<li>After preparing libraries, measure DNA concentration using fluorometer.</li>
	<li>Measure DNA size using electrophoresis.</li>
	<li>Perform sequencing on any platform. 
	NOTE: Sequencing at least 10 M reads per sample is recommended. This depth is equivalent to a read approximately every 300 bp (for ~3 Gb size genomes), and is sufficient for ToR measurements at a resolution of 50 - 100 kb. Increasing the depth will result in a decrease in the size of the windows and thus will enable increasing resolution with higher certainty. Paired end sequencing is not necessary in this protocol since only coverage information is collected. It, however, may help in resolving the location of reads that contain repetitive sequences.</li>
</ol>
8. Analysis
NOTE: Data analysis is based on the method used by A. Koren et al.19.
<ol>
	<li>Map sequencing data to the corresponding genome using bowtie2 or any reliable short read aligner. Define varying-size, equal-coverage chromosomal windows as segments covered by 200 reads in the G1 fraction and count S phase reads in the same windows.</li>
	<li>Calculate the S/G1 ratio for each window. This should generate a map with large fluctuations in the S/G1 ratio along the genome (Figure 3). A good control for the reliability of the ToR measurements is to compare this map to the G1/G1 ratio (from two separate measurements of G1) which should be much flatter.</li>
	<li>Normalize data to 0 mean and 1 SD by subtracting from each value the average value of all windows (excluding the X chromosome) and dividing the results by the standard deviation of the S/G1 of all windows. This is done in order to convert to z scores and enable comparison between different experiments.</li>
	<li>Remove all gap regions listed by the UCSC genome browser as well as each remaining inter gap fragment containing fewer than 15 data windows.</li>
	<li>Smooth the remaining fragments with a cubic smoothing spline via the Matlab function csaps with a parameter of 10&#8722;16 and interpolate at set points every 100 kb. 
	NOTE: Parameters of smoothing and interpolation should be adjusted based on the depth of the data. Other suitable smoothing methods and functions exist and can be used.</li>
	<li>After visually confirming the reliability of each replicate, merge all the reads and calculate a deeper resolution profile by performing the same process described above on this data.</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+replication+timing+and+gene+expression+during+lineage+specification+of+human+pluripotent+stem+cells.">Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells.</a> Genome Res. 25 (8), 1091-1103 (2015).</li><li>Ryba, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+developmental+control+of+replication-timing+domains+in+pediatric+acute+lymphoblastic+leukemia.">Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia.</a> Genome Res. 22 (10), 1833-1844 (2012).</li><li>Farkash-Amar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+organization+of+replication+time+zones+of+the+mouse+genome.">Global organization of replication time zones of the mouse genome.</a> Genome Res. 18 (10), 1562-1570 (2008).</li><li>Koren, A., McCarroll, S. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+timing+and+selection+shape+the+landscape+of+nucleotide+variation+in+cancer+genomes.">DNA replication timing and selection shape the landscape of nucleotide variation in cancer genomes.</a> Nat Commun. 3, 1004 (2012).</li><li>Liu, L., De, S., Michor, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+timing+and+higher-order+nuclear+organization+determine+single-nucleotide+substitution+patterns+in+cancer+genomes.">DNA replication timing and higher-order nuclear organization determine single-nucleotide substitution patterns in cancer genomes.</a> Nat Commun. 4, 1502 (2013).</li><li>Goren, A., Cedar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicating+by+the+clock.">Replicating by the clock.</a> Nat Rev Mol Cell Biol. 4 (1), 25-32 (2003).</li><li>Selig, S., Okumura, K., Ward, D. 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No conflicts of interest declared.

CNR-ToR can be performed in principle on any eukaryotic proliferating cell population that can be divided by FACS to S and G1 phases (reviewed by Rhind N. and Gilbert DM20). The method described here has been adjusted to mammalian cells with a genome size of ~3 Gb such as human and mouse. Small changes in the CNR-ToR protocol (in cell preparation and sequencing depth) are needed, in order to adjust it to other eukaryotes. Attention must be paid to the collection of sufficient amounts of S phase ce...

Mammalian DNA replication is tightly regulated to ensure the precise replication of each chromosome exactly once during the cell cycle. Replication occurs according to a highly regulated order &#8212; multiple large genomic regions (~Mb) replicate at the beginning of S phase (early replicating domains) whereas other genomic regions replicate later at middle or late S phase (middle and late replicating domains)1. Most of the genome replicates at the same time in all tissues (constitutive ToR domains), whereas 30% - 50% of the genome, changes its ToR between tissues2, during differentiation3,4 and to a lesser extent also during cancer transformation5. Moreover, certain genomic regions replicate asynchronously6,7,8, namely there is a difference in the ToR between the two alleles.
ToR correlates with many genomic and epigenomic features including transcription levels, GC content, chromatin state, gene density, etc.1,9. ToR is also associated with mutation rates and types10,11 and therefore unsurprisingly, perturbations of the replication program are linked to cancer12,13. The causal relationship between ToR and chromatin structure is not yet understood. It is possible that open chromatin facilitates early replication. However, an alternative model suggests that the chromatin is assembled during replication and the different chromatin regulators present at the beginning and end of S phase lead to differential packaging of early and late replicating regions1,14. We have recently shown that the ToR shapes the GC content by affecting the type of mutations that occur in different genomic regions11.
Fluorescence in situ hybridization (FISH) is the main method for measuring ToR at individual loci. It is performed simply by counting the percentage of S phase cells that exhibit single FISH signals vs. the percentage of doublets for a given allele15,16. An alternative method, consists of pulse labeling the DNA with BrdU, sorting cells according to their DNA content to multiple time points along S, immunoprecipitating DNA containing BrdU, and checking the abundance of precipitated DNA with qPCR17.
Genomic ToR mapping can be achieved by two methods. The first method is a genomic version of the BrdU-IP based method described above, in which the quantification of the amount of precipitated DNA in each fraction is done simultaneously for the entire genome through hybridization to microarrays or by deep sequencing. The second method, CNR-ToR, is based on measuring the copy number of each genomic region of S phase cells and normalizing by the DNA content in G1 cells. In this method, cells are sorted by FACS into non-replicating (G1 phase) and replicating (S phase) groups (Figure 1). Cells in G1 have the same copy number in all genomic regions and thus their DNA content should be the same. On the other hand, the DNA copy number in S depends on the ToR, since early replicating regions underwent replication in most cells and therefore their DNA content is doubled, whereas late replicating regions have not replicated yet in most cells and therefore their DNA content will be similar to that of G1 cells. Hence the S to G1 ratio of DNA content is indicative of the ToR. The amount of DNA for each genomic region is measured either by hybridization to microarrays or by deep sequencing2,8. The advantages of the CNR-ToR method will be further discussed.
This paper describes the CNR-ToR method for genomic ToR mapping as described in Figure 2. The paper discusses the fine details of the entire process from collecting cells until the basic analysis of the results and the creation of genomic ToR maps. The protocol described in this paper has been successfully performed on various cell types grown in culture. Future improvements of this protocol can lead to the mapping of the ToR in vivo and in rare cell types.

<table><tbody><tr><td>PBS</td><td>BI (Biological Industries)</td><td>02-023-1A</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>BI (Biological Industries)</td><td>03-052-1B</td><td></td></tr><tr><td>15 mL conical tube</td><td>Corning</td><td>430790</td><td></td></tr><tr><td>5 mL Polystyrene round Bottom tube with cell strainer cap&amp;nbsp;</td><td>BD-Falcon</td><td>352235</td><td></td></tr><tr><td>Ethanol</td><td>Gadot</td><td>64-17-5</td><td></td></tr><tr><td>RNAse-A 10 mg/mL</td><td>Sigma</td><td>R4875</td><td></td></tr><tr><td>Propidiom iodide 1 mg/mL</td><td>Sigma</td><td>P4170</td><td></td></tr><tr><td>Parafilm</td><td>Parafilm</td><td>PM-996</td><td></td></tr><tr><td>1.5 mL DNA LoBind Eppendorf tubes&amp;nbsp;</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>1.7 mL MaxyClear tube&amp;nbsp;</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>magnetic beads - Agencourt AMPure XP&amp;nbsp;</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Ultrasonicator</td><td>Covaris</td><td>M-series&amp;nbsp; -530092</td><td></td></tr><tr><td>50 &amp;micro;L microTUBE AFA Fiber Screw-Cap 6 x 16 mm</td><td>Covaris</td><td>520096</td><td></td></tr><tr><td>Qubit fluorometer</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Qubit dsDNA High Sensitivity (HS) Assay Kit</td><td>Invitrogen</td><td>Q32854</td><td></td></tr><tr><td>Electrophoresis 2200 Tape station system</td><td>Agilent</td><td>D1000 ScreenTape</td><td></td></tr><tr><td>Seqeuncing - Illumina NextSeq system</td><td>Illumina</td><td>SY-415-1001</td><td></td></tr><tr><td>Dneasy kit for DNA purification</td><td>Qiagen</td><td>69504</td><td></td></tr><tr><td>PureProteom Magnetic Stand</td><td>Millipore</td><td>LSKMAGS08</td><td></td></tr><tr><td>Anti-BrdU/FITC</td><td>DAKO</td><td>F7210</td><td></td></tr><tr><td>FACS sorter</td><td>BD</td><td>FACSARIA III</td><td></td></tr><tr><td>FACS software</td><td>BD</td><td>FACSDiva v 8.0.1</td><td></td></tr></tbody></table>

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.

A typical ToR map is shown in Figure 3 for mouse embryonic fibroblasts (MEFs). This figure demonstrates the analysis process since it shows both the dots, which are the normalized S/G1 ratio for individual windows (step 8.3), as well as the line which results from the cubic smoothing and interpolation (step 8.5).
Such maps capture the organization of the replication program, which is a patchwork of two types of ...

Watch this Scientific Journal Video about Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement at JoVE.com

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

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 ...

genome-wide-determination-mammalian-replication-timing-dna-content

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

8.4K Views.  Hebrew University of Jerusalem. 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.

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

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

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 ...

Biology

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. ...

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Cancer Research

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

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.

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 ...

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 ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome segregation. As DNA synthesis defects underlie chromosome rearrangements, monitoring DNA replication has become essential to understand the basis of genome instability. Saccharomyces cerevisiae is a classical model to study cell cycle regulation, but key DNA replication parameters, such as the fraction of cells in the S phase or the S-phase duration, are still difficult to determine. This protocol uses short and non-toxic pulses of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, in engineered TK-hENT1 yeast cells, followed by its detection by Click reaction to allow the visualization and quantification of DNA replication with high spatial and temporal resolution at both the single-cell and population levels by microscopy and flow cytometry. This method may identify previously overlooked defects in the S phase and cell cycle progression of yeast mutants, thereby allowing the characterization of new players essential for ensuring genome stability.

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

We describe two complementary protocols to accurately determine S-phase duration in S. cerevisiae using EdU, a thymidine analog, which is incorporated in vivo and detected using Click chemistry by microscopy and flow cytometry. It allows for the easy characterization of the duration of DNA replication and overlooked replication defects in mutants.

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome ...

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

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G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

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

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

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

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

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae