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>
	<li>Bell, S. P., Labib, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+duplication+in+Saccharomyces+cerevisiae.">Chromosome duplication in Saccharomyces cerevisiae.</a> Genetics. 203 (3), 1027-1067 (2016).</li><li>Kitamura, E., Blow, J. J., Tanaka, T. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+reveals+replication+of+individual+replicons+in+eukaryotic+replication+factories.">Live-cell imaging reveals replication of individual replicons in eukaryotic replication factories.</a> Cell. 125 (7), 1297-1308 (2006).</li><li>Marston, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+segregation+in+budding+yeast:+Sister+chromatid+cohesion+and+related+mechanisms.">Chromosome segregation in budding yeast: Sister chromatid cohesion and related mechanisms.</a> Genetics. 196 (1), 31-63 (2014).</li><li>Lengronne, A., 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=The+yeast+CDK+inhibitor+Sic1+prevents+genomic+instability+by+promoting+replication+origin+licensing+in+late+G1.">The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G1.</a> Molecular Cell. 9 (5), 1067-1078 (2002).</li><li>Tanaka, S., Diffley, J. F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulated+G+1-cyclin+expression+induces+genomic+instability+by+preventing+efficient+pre-RC+formation.">Deregulated G 1-cyclin expression induces genomic instability by preventing efficient pre-RC formation.</a> Genes &amp; Development. 16 (20), 2639-2649 (2002).</li><li>Teixeira, L. K., 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=Cyclin+E+deregulation+promotes+loss+of+specific+genomic+regions.">Cyclin E deregulation promotes loss of specific genomic regions.</a> Current Biology. 25 (10), 1327-1333 (2015).</li><li>Slater, M. L., Sharrow, S. O., Gart, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+of+Saccharomyces+cerevisiae+in+populations+growing+at+different+rates.">Cell cycle of Saccharomyces cerevisiae in populations growing at different rates.</a> Proceedings of the National Academy of Sciences of the United States of America. 74 (9), 3850-3854 (1977).</li><li>McNeil, J. B., Friesen, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+herpes+simplex+virus+thymidine+kinase+gene+in+Saccharomyces+cerevisiae.">Expression of the herpes simplex virus thymidine kinase gene in Saccharomyces cerevisiae.</a> Molecular and General Genetics. 184 (3), 386-393 (1981).</li><li>Vernis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+an+efficient+thymidine+salvage+pathway+in+Saccharomyces+cerevisiae.">Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae.</a> Nucleic Acids Research. 31 (19), 120 (2003).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (7), 2415-2420 (2008).</li><li>Bruschi, C. V., McMillan, J. N., Coglievina, M., Esposito, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+instability+of+yeast+cdc6-1/cdc6-1+mutants+involves+chromosome+structure+and+recombination.">The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination.</a> Molecular and General Genetics. 249 (1), 8-18 (1995).</li><li>Feng, L., Wang, B., Wu, L., Jong, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+control+of+Mcm5+interaction+with+chromatin+in+cdc6-1+mutated+in+CDC-NTP+motif.">Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif.</a> DNA and Cell Biology. 19 (7), 447-457 (2000).</li><li>Saner, N., 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=Stochastic+association+of+neighboring+replicons+creates+replication+factories+in+budding+yeast.">Stochastic association of neighboring replicons creates replication factories in budding yeast.</a> Journal of Cell Biology. 202 (7), 1001-1012 (2013).</li><li>Pasero, P., Braguglia, D., Gasser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORC-dependent+and+origin-specific+initiation+of+DNA+replication+at+defined+foci+in+isolated+yeast+nuclei.">ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei.</a> Genes &amp; Development. 11 (12), 1504-1518 (1997).</li><li>Lengronne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+S+phase+progression+globally+and+locally+using+BrdU+incorporation+in+TK++yeast+strains.">Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains.</a> Nucleic Acids Research. 29 (7), 1433-1442 (2001).</li><li>Magiera, M. 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 ...

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