Name: determination of s-phase duration using 5-ethynyl-2'-deoxyuridine incorporation in saccharomyces cerevisiae
Uploaded: 2022-10-21T13:30:00
Description: Watch this Scientific Journal Video about Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae at JoVE.com

The authors wish to acknowledge Agence Nationale de la Recherche (ANR) and Association pour la Recherche sur le Cancer (ARC) for the PhD fellowships to J.d.D.B.T. and the Agence Nationale pour la Recherche (ANR) for financial support (grant ANR-18-CE12-0018-01). Cytometry and microscopy were performed at the Montpellier MRI BioCampus imaging facility.

1. S. cerevisiae cell culture
NOTE: The yeast strains used are listed in Table 1
NOTE: The S-phase duration can be monitored in different ways. Depending on the question to be addressed, the cells can be grown asynchronously or synchronously following G1 arrest.
<ol>
	<li>From asynchronously growing S. cerevisiae cells 
	NOTE: This method allows for the determination of the percentage of cells in ...

<ol>
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M., Gueydon, E., Schwob, E. <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+and+spindle+checkpoints+cooperate+during+S+phase+to+delay+mitosis+and+preserve+genome+integrity.">DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity.</a> The Journal of Cell Biology. 204 (2), 165-175 (2014).</li><li>Talarek, N., Petit, J., Gueydon, E., Schwob, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EdU+incorporation+for+FACS+and+microscopy+analysis+of+DNA+replication+in+budding+yeast.">EdU incorporation for FACS and microscopy analysis of DNA replication in budding yeast.</a> Methods in Molecular Biology. 1300, 105-112 (2015).</li><li>Ivanova, 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=Budding+yeast+complete+DNA+synthesis+after+chromosome+segregation+begins.">Budding yeast complete DNA synthesis after chromosome segregation begins.</a> Nature Communications. 11 (1), 2267 (2020).</li></ol>

Yeast is a prime model organism for cell cycle studies, yet the characterization of its S phase has long been hampered by its inability to incorporate exogenous nucleosides, such as BrdU, which are used as tracers of DNA replication. Equipping yeast with a high expression of herpes simplex thymidine kinase (TK) and the addition of a human nucleoside transporter (hENT) has largely solved this problem15,16. EdU is more versatile than BrdU as its detection with smal...

Genome stability through mitotic division is ensured by the transmission of a complete and equal set of chromosomes to the two produced cell progenies. This relies on the accurate completion of a series of events occurring in a given time in each phase of the cell cycle. In G1, the replication origins are licensed upon the recruitment of several licensing factors, including Cdc61. In the S phase, whole-genome duplication is initiated from multiple active replication origins and performed by replication machineries that gather in microscopically visible foci named replication factories2. In the M phase, duplicated sister chromatids are attached and bioriented on the mitotic spindle to allow their segregation to the opposite poles of the mitotic cell3. The regulation, proper completion, and duration of each phase are key to ensure genome stability. Indeed, premature exit from any of these phases leads to genome instability. For instance, a shorter G1 induced by deletion of the budding yeast CDK inhibitor Sic1 or by the overexpression of G1 cyclins will alter the subsequent S phase4,5,6. Consequently, these deregulations, associated or not with replication stress, result in chromosome breaks, rearrangements, and mis-segregation4,5,6. Therefore, monitoring the duration of the S phase and, more broadly, the duration of the other phases of the cell cycle may be crucial to identify the defects occurring in different mutants and in different stressful conditions.
A traditional method for measuring cell cycle phase duration includes simple DNA content flow cytometry (Figure 1A) and relies on a fitting algorithm (available in most cytometry software) used to separate the population into G1, S, and G2 + M phase fractions from the 1C and 2C peaks. The fractions are then multiplied by the population doubling time7. However, this method gives only estimated values, requires a homogeneous cell size distribution within a given fraction, and is not applicable to synchronized cultures. To study the S-phase duration in mammalian cells, several thymidine analogs have been developed and widely used, including EdU. Their uptake from the extracellular medium and phosphorylation by thymidine kinase (hereafter referred to as TK) make them available for DNA polymerases to incorporate them at sites of DNA synthesis (replication, recombination, repair). To bypass the absence of the TK gene in Saccharomyces cerevisiae cells, yeast strains have been engineered to allow stable and constitutive expression of the herpes simplex virus TK8 and the human equilibrative nucleoside transporter (hENT1)9. Once incorporated into DNA, EdU is detected via the selective Click reaction, which chemically couples its alkyne moiety to azide-modified fluorochromes10.
This paper provides two optimized comprehensive protocols to pulse-label asynchronous and synchronous TK-hENT1 engineered cells with EdU in order to precisely visualize and measure DNA replication duration and dynamics, as well as the duration of the other phases of the cell cycle, with high spatial and temporal resolution at both the single-cell and population levels by microscopy and flow cytometry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#945;-factor&#160;</td>
			<td>Genescript</td>
			<td>RP01002</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Euromedex</td>
			<td>04-100--812-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper sulfate</td>
			<td>Sigma</td>
			<td>C1297</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-sulfo-Cyanine5 azide (Cy5 azide)</td>
			<td>Interchim</td>
			<td>FP-JV6320</td>
			<td>Alternative to Alexa647-Azide</td>
		</tr>
		<tr>
			<td>Dy-530 azide</td>
			<td>Dyomics</td>
			<td>&#160;530-10</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU (5-ethynyl-2&#8217;-deoxyuridine)</td>
			<td>Carbosynth</td>
			<td>NE08701</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Carlo Erba reagents</td>
			<td>P013A10D16</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>L- ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Euromedex</td>
			<td>EU0090</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase</td>
			<td>SIGMA</td>
			<td>R5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sytox Green</td>
			<td>Invitrogen</td>
			<td>S-7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>OLS</td>
			<td>CASY</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Agilent</td>
			<td>NovoSampler Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Infors</td>
			<td>444-4230</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Shaking water bath</td>
			<td>Julabo</td>
			<td>SW22</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics</td>
			<td>Vibra cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field microscopy</td>
			<td>Leica</td>
			<td>THUNDER Imager</td>
			<td>or equivalent</td>
		</tr>
	</tbody>
</table>

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.

To determine the S-phase duration and, more broadly, the duration of G1 and G2 + M (protocol step 1.1), S. cerevisiae W303 wild-type cells (WT, Table 1) were grown asynchronously in SC medium for 7 h. Every hour, the cell concentration was monitored to determine the doubling time (Figure 2B). In these growth conditions, the calculated doubling time was 120 min &#177; 13 min at 25 &#730;C (Table 2). When the cells were in the ex...

Watch this Scientific Journal Video about Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae at JoVE.com

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.

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 ...

This protocol allows a precise determination of S-phase duration in synchronized budding yeast cells. It is a quick, easy, and reproducible method. In addition, it is sensitive enough to detect mild synthesis defects undetected by classical protocols.This method will be useful for many researchers working on cell cycle regulation in budding yeast. Demonstrating this protocol will be Nicolas Talarek, a senior researcher, and Juan De Dios Barba Tena, a PhD student in the lab. Inoculate Saccharomyces cerevisiae cells in 10 milliliters of SC medium at a low cell concentration for an overnight culture at 30 degree Celsius with orbital agitation at 130 rotations per minute.The following day, dilute the cells in 20 milliliters of fresh SC medium at a final concentration of two to three times 10 to the six cells per milliliter. At 40 microliters of one milligram per milliliter alpha factor diluted in water. Then culture the cells at 30 degree Celsius, with orbital agitation at 130 rotations per minute for one hour.Repeat this step once. Visualize the cells under a light microscope to monitor G1 arrest. Proceed if more than 90%of the cells display a shmoo, and the others are rounded unbutted cells.Centrifuge the samples for three minutes at 1, 500 G.Then discard the supernatant with the vacuum pipette and resuspend the cells in 20 milliliters of SC medium. Repeat this step once. Collect one milliliter of cells twice every five minutes and proceed to EDU labeling.Add 400 microliters of the alpha factor 30 minutes after the release. Transfer one milliliter of the cell culture to a two milliliter microfuge tube containing one microliter of 10 millimolar EDU, mixed well by inversion. To discriminate the EDU positive cells from the EDU negative cells on a Pi EDU bivariant facts, transfer another one milliliter of cell culture to a two milliliter microfuge tube containing one microliter of DMSO.Incubate for three to five minutes at 30 degree Celsius under agitation in a shaking water bath. Stop the reaction by adding 100 microliters of 100%ethanol. Pellet the cells for two minutes at 10, 000 G in a microfuge.Remove the supernatant using a vacuum pipette. Resuspend the cell pellet in 500 microliters of 70%ethanol, and mix well by vortexing. Leave the samples at room temperature for at least one hour at 20 tilts per minute on a variable speed rocker to permeablizie the cells.Pellet the cells for two minutes at 10, 000 G in a microcentrifuge and discard the supernatant with a vacuum pipette. Wash the cells twice with 500 microliters of 10%ethanol and PBS. Pellet the cells for two minutes at 10, 000 G in a microfuge and discard the supernatant with a vacuum pipette.Resuspend the pellet in 200 microliters of PBS containing RNase A and Proteinase K.Incubate from one to two hours at 50 degree Celsius with occasional shaking, or overnight at 37 degree Celsius. Pellet the cells for two minutes at 10, 000 G in a micro centrifuge and discard the supernatant with a vacuum pipette. Wash the cells with 500 microliters of PBS.Then pellet the cells and discard the supernatant as demonstrated earlier. Resuspend the cell pellet in 200 microliters of PBS with 1%BSA. Incubate for 30 minutes at room temperature.Then pellet the cells, discard the supernatant as demonstrated earlier, and resuspend the pellet in 300 microliters of PBS containing 1%BSA. Distribute the cells into two, 1.5 milliliter microcentrifuge tubes. The first one should consist of 200 microliters of cell culture for the Click reaction, and the second tube should contain 100 microliters of cell culture for the sytox green staining.Then, pellet the cells and discard the supernatant as demonstrated earlier. For sytox green staining, resuspend the cell pellet in 100 microliters of PBS. Then depending on the cell concentration, transfer 10 to 30 microliters to a flow cytometer tube containing 300 microliters of sytox green mixture.Sonicate the samples twice for two seconds at an amplitude of 40 to 50%Leave in the dark until processing the samples on a flow cytometer. For the Click reaction, resuspend the cell pellet with 40 microliters of freshly prepared Azide dye buffer. Incubate at room temperature in the dark for 60 minutes.Pellet the cells and discard the supernatant as demonstrated earlier. Then wash the cells three times with 300 microliters of 10%ethanol in PBS. Next resuspend the cells in 100 microliters of 50 micrograms per milliliter propidium iodide in PBS and leave for 10 minutes in the dark.Depending on the cell concentration, transfer 10 to 30 microliters of the cell suspension to a flow cytometer tube containing 300 microliters of 50 millimolar Tris HCl, pH 7.5. Sonicate twice for two seconds at an amplitude of 40 to 50. Leave the samples in the dark until processing them on a cytometer.Read the sytox green samples using an excitation blue laser at 488 nanometers, and a 530 by 30 band pass filter. Read the Bavaria Pi EDU samples on a dot plot using an excitation blue laser at 488 nanometers and a 615 by 20 band pass filter for the Pi X-axis, and a red excitation laser at 640 nanometers, a 660 by 20 band pass filter for the Y-axis. Three populations of cells were observed in the bivariant EDU Pi cytometer analysis.At 25 degree Celsius, approximately 27%of the cell population was in the G1 phase, 29%was in the S phase, and about 44%was in G2 plus M phase. In synchronized cells, the duration of the S phase may be about 25 minutes based on the time when the DNA content changed from one C to two C on the sytox green flow cytometer profile. EDU pulse labeling accurately determined the S phase duration.15 minutes after the release, a fraction of EDU positive cells was detected, indicating the start of the S phase. The progression through the S phase was seen by the cell cloud moving upward and rightward in the Bavaria Pi EDU graph. Finally, at 35 minutes from the release, a fraction of cells was EDU negative, but with twice the amount of DNA indicating that the S phase ended and the cells were in the G2 plus M phase.Despite the high synchrony observed, some cells finished the S phase 60 minutes after the release and the S phase was complete for the whole population approximately 65 minutes after the release. It is crucial to have a perfect synchronization and to prevent cells entering the second S phase. Cells can be imaged with a microscope where nuclear DNA replication foresight can be monitored and quantified.This technique will help to identify overlook mutants that have mild S phase defects, as well as cell cycle duration defects.

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