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 the S phase in an asynchronously growing cell population. By determining the doubling time, the duration of the S phase (and the other phases) can be extrapolated.
	<ol>
		<li>Inoculate S. cerevisiae cells in 10 mL of synthetic complete (SC) medium at a low cell concentration (5 &#215; 104 cells/mL) for an overnight culture at 30 &#730;C with orbital agitation at 130 rpm. 
		NOTE: The concentration is measured with a cell counter. To efficiently calculate the doubling times, it is recommended to inoculate the culture from an overnight culture that is still in the exponential phase (i.e., ideally below 2 &#215; 107 cells/mL). Growing cells in rich medium (YPD) is not recommended, as EdU detection is not efficient.</li>
		<li>The following day, dilute the cells in 20 mL of fresh SC medium at the final concentration of 5 &#215; 105 cells/mL.</li>
		<li>Culture the cells at 30 &#730;C in a shaking water bath with horizontal shaking at 120 rpm.</li>
		<li>Measure the cell concentration every hour until it reaches 1 &#215; 107 cells/mL. 
		NOTE: This step allows for making a graphical representation of the cell concentration increase over time. The formula used to calculate the doubling times is explained in the legend of Table 2.</li>
		<li>Proceed in parallel to step 2 for EdU labeling when the cell concentration is about 2 &#215; 106-5 &#215; 106 cells/mL.</li>
	</ol>
	</li>
	<li>From G1-synchronized S. cerevisiae cells 
	NOTE: This method allows for the determination of when the S phase starts and finishes by means of flow cytometry and/or microscopy analyses.
	<ol>
		<li>Inoculate S. cerevisiae cells in 10 mL of SC medium at a low cell concentration (5 &#215; 104 cells/mL) for an overnight culture at 30 &#730;C with orbital agitation at 130 rpm. 
		NOTE: See the notes after step 1.1.1.</li>
		<li>The following day, dilute the cells in 20 mL of fresh SC medium at a final concentration of 2 &#215; 106-3 &#215; 106 cells/mL.</li>
		<li>Add 40 &#181;L of 1 mg/mL &#945;-factor diluted in water.</li>
		<li>Culture the cells at 30 &#730;C with orbital agitation at 130 rpm for 1 h.</li>
		<li>Add again 40 &#181;L of 1 mg/mL &#945;-factor diluted in water.</li>
		<li>Culture the cells at 30 &#730;C with orbital agitation at 130 rpm for 1 h.</li>
		<li>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, unbudded cells. 
		NOTE: Depending on the background used, it is recommended to sonicate the cells before shmoo visualization. For the W303 background, sonicate 2x, for 2 s each time, at an amplitude of 40-50.</li>
		<li>Centrifuge for 3 min at 1,500 &#215; g. Discard the supernatant with a vacuum pipette.</li>
		<li>Resuspend the cells in 20 mL of SC medium.</li>
		<li>Repeat steps 1.2.8-1.2.9 once. 
		NOTE: The &#945;-factor is washed away with these steps, and the cells are released in the cell cycle. Alternatively, the &#945;-factor may be washed away by filtering the yeast cells with a 1.2 &#181;m nitrocellulose filter using a funnel set on a side-arm flask connected to a vacuum pump.</li>
		<li>Collect 1 mL of cells 2x every 5 min and proceed to step 2 for EdU labeling. 
		NOTE: Pulse-label the cells from only one of the two tubes with EdU. The non-pulse-labeled cells are used to distinguish EdU-positive from EdU-negative cells on a bivariate propidium iodide (PI)-EdU graph.</li>
		<li>Add 400 &#181;L of 1 mg/mL &#945;-factor diluted in water 30 min after the release. 
		&#8203;NOTE: This high dose of &#945;-factor is required to arrest the cells in the G1 phase of the next cell cycle and to prevent the cells from re-entering the subsequent S phase.</li>
	</ol>
	</li>
</ol>
2. EdU labeling
<ol>
	<li>Transfer 1 mL of cell culture to a 2.0 mL microfuge tube containing 1 &#181;L of 10 mM EdU. Mix well by inversion. 
	NOTE: To discriminate the EdU-positive cells from the EdU-negative cells on a PI-EdU bivariate FACS, transfer another 1 mL of cell culture to a 2.0 mL microfuge tube containing 1 &#181;L of DMSO.</li>
	<li>Incubate for 3-5 min at 30 &#730;C under agitation in a shaking water bath. 
	NOTE: Three minutes are sufficient for EdU detection with a microscope; 5 min are required for optimal EdU detection on a flow cytometer.</li>
	<li>Stop the reaction with the addition of 100 &#181;L of 100% ethanol.
	<ol>
		<li>Stop the reaction with the addition of 100 &#181;L of 20% paraformaldehyde if cell size measurement is required. 
		NOTE: If the nuclear architecture of the mitotic cells is to be kept intact for further analyses following the Click reaction, we recommend fixing cells with 2% paraformaldehyde at room temperature (RT) rather than putting the cells on ice, since the latter causes microtubule depolymerization.</li>
		<li>Leave the cells for 20 min at RT under mild agitation on a variable speed rocker at 20 tilts/min to fix the cells before the addition of 100 &#181;L of 100% ethanol.</li>
	</ol>
	</li>
</ol>
3. Cell fixation and permeabilization
<ol>
	<li>Pellet the cells for 2 min at 10,000 &#215; g in a microfuge. Remove the supernatant using a vacuum pipette.</li>
	<li>Resuspend the cell pellet in 500 &#181;L of 70% ethanol. Mix well by vortexing.</li>
	<li>Leave for &#8805;1 h at RT at 20 tilts/min on a variable speed rocker to permeabilize the cells. 
	&#8203;NOTE: Cells grown in SC medium do not pellet well as they tend to stick to the microfuge walls. The addition of ethanol improves pelleting and reduces cell loss. The cells may be stored at 4 &#730;C overnight or for longer periods at &#8722;20 &#730;C.</li>
	<li>Pellet the cells for 2 min at 10,000 &#215; g in a microcentrifuge. Discard the supernatant with a vacuum pipette.</li>
	<li>Wash the cells 2x with 500 &#181;L of 10% ethanol in PBS. 
	&#8203;NOTE: The washes are crucial to remove unincorporated EdU from the cells.</li>
</ol>
4. Click-it reaction
<ol>
	<li>For cytometry analysis
	<ol>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microfuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Resuspend the pellet in 200 &#181;L of PBS containing 0.1 mg/mL RNase A and 0.2 mg/mL proteinase K.</li>
		<li>Incubate for 1-2 h at 50 &#176;C with occasional shaking (or overnight at 37 &#176;C).</li>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microcentrifuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Wash the cells with 500 &#181;L of PBS.</li>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microfuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Resuspend the cell pellet in 200 &#181;L of PBS + 1% bovine serum albumin (BSA). Incubate for 30 min at RT. 
		NOTE: Longer times are not necessary and are even detrimental to the efficiency of Click reactions.</li>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microcentrifuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Resuspend the pellet in 300 &#181;L of PBS + 1% BSA.</li>
		<li>Distribute the cells between two tubes: 200 &#181;L in a 1.5 mL microcentrifuge tube for the Click reaction and 100 &#181;L in another 1.5 mL microcentrifuge tube for Sytox Green staining.</li>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microcentrifuge. Discard the supernatant with a vacuum pipette.</li>
		<li>For Sytox Green staining 
		NOTE: We highly recommend taking an aliquot for Sytox Green staining (without Click) in order to obtain high-quality DNA content reference profiles. Indeed, the Click reaction strongly quenches intercalant fluorescence, including the Sytox Green and PI fluorescence. Consequently, the Click reaction can distort the reading of DNA content.
		<ol>
			<li>Resuspend the cell pellet in 100 &#181;L of PBS.</li>
			<li>Transfer 10-30 &#181;L (depending on the cell concentration) to a flow cytometer tube containing 300 &#181;L of 50 mM Tris-HCl, pH 7.5, and 0.5 &#181;M Sytox Green.</li>
			<li>Sonicate 2x, for 2 s each time, at an amplitude of 40-50.</li>
			<li>Leave in the dark until processing the samples on a flow cytometer. 
			NOTE: The cells can be kept at this stage at 4 &#730;C for a few days.</li>
		</ol>
		</li>
		<li>For the Click reaction
		<ol>
			<li>Prepare fresh Azide Dye buffer by mixing the reagents in the following order (quantity for one tube): 36 &#181;L of PBS, 2 &#181;L of 0.2 M CuSO4, 0.2 &#181;L of 2 mM Cy5 Azide, and 2 &#181;L of 1 M ascorbic acid. 
			NOTE: It is possible to prepare a master mix of the Azide Dye buffer. The reagents have to be mixed in the same order as that mentioned above.</li>
			<li>Resuspend the cell pellet with 40 &#181;L of freshly made Azide Dye mix. Incubate at RT in the dark for 60 min.</li>
			<li>Pellet the cells for 2 min at 10,000 &#215; g in a microcentrifuge. Discard the supernatant with a vacuum pipette.</li>
			<li>Wash the cells 3x with 300 &#181;L of 10% ethanol in PBS. 
			NOTE: The washes are crucial to eliminate all the soluble EdU-Cy5 azide.</li>
			<li>Resuspend the cells in 100 &#181;L of 50 &#181;g/mL PI in PBS. Leave for 10 min in the dark.</li>
			<li>Transfer 10-30 &#181;L of the cell suspension (depending on the cell concentration) to a flow cytometer tube containing 300 &#181;L of 50 mM Tris-HCl, pH 7.5.</li>
			<li>Sonicate 2x, for 2 s each time, at an amplitude of 40-50.</li>
			<li>Leave in the dark until processing the samples on a cytometer. 
			NOTE: The cells can be kept at this stage at 4 &#730;C for a few days.</li>
		</ol>
		</li>
		<li>Read the Sytox Green samples using an excitation blue laser at 488 nm and a 530/30 BP filter. See Figure 1A for typical results. Read the bivariate PI-EdU samples on a dot plot using an excitation blue laser at 488 nm and a 615/20 BP filter for the PI (x-axis) and an excitation red laser at 640 nm and a 660/20 BP filter (y-axis). See Figure 1B for typical results. 
		NOTE: Figure 1C represents the typical PI-EdU bivariate FACS result for EdU-negative cells. It allows for the discrimination of the EdU-negative cells from the EdU-positive cells.</li>
	</ol>
	</li>
	<li>For microscopy analysis
	<ol>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microcentrifuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Resuspend the pellet in 200 &#181;L of PBS + 1% BSA. Incubate for 30 min at RT.</li>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microfuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Prepare fresh Azide Dye buffer by mixing the reagents in the following order (quantity for one tube): 36 &#181;L of PBS, 2 &#181;L of 0.2 M CuSO4, 0.2 &#181;L of 2 mM Dy-530 azide, 2 &#181;L of 1 M ascorbic acid. 
		NOTE: A master mix of the Azide Dye buffer can be prepared fresh by mixing the reagents in the aforementioned order.</li>
		<li>Resuspend the pellet with 40 &#181;L of freshly made Azide Dye buffer. Incubate at RT in the dark for 60 min.</li>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microfuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Wash the cells 2x with 300 &#181;L of 10% ethanol in PBS. 
		NOTE: The washes are crucial to eliminate all soluble Dy-530 azide.</li>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microcentrifuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Resuspend the cells in 100 &#181;L of 0.5 &#181;g/mL 4&#39;,6-diamidino-2-phenylindole (DAPI) in PBS. Leave for 30 min in the dark at room temperature.</li>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microcentrifuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Wash with 300 &#181;L of PBS to remove excess DAPI.</li>
		<li>Pellet the cells for 2 min at 10,000 &#215; g in a microfuge. Discard the supernatant with a vacuum pipette.</li>
		<li>Resuspend the pellet with 10-50 &#181;L of PBS depending on the cell concentration.</li>
		<li>Sonicate 2x, for 2 s each time, at an amplitude of 40-50. 
		NOTE: The cells can be kept at this stage at 4 &#730;C for a few days.</li>
		<li>Pipette 1.7 &#181;L of the cells onto a glass microscope slide and cover with a clean coverslip.</li>
		<li>Immediately observe under a fluorescence microscope with DAPI and TexasRed or Cy3 filters.</li>
	</ol>
	</li>
</ol>

<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>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>&amp;alpha;-factor&amp;nbsp;</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>&amp;nbsp;530-10</td><td></td></tr><tr><td>EdU (5-ethynyl-2&amp;rsquo;-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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 ...

determination-s-phase-duration-using-5-ethynyl-2-deoxyuridine

Research

JoVE Journal

Biology

1.3K Views.  Université de Montpellier, CNRS. 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.

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

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 ...

Genetics

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Use of the Pyrimidine Analog, 5-Iodo-2&prime;-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the cell cycle is a feature of a number of diseases, including cancer. Study of the cell cycle necessitates the ability to define the number of cells in each portion of cell cycle progression as well as to clearly delineate between each cell cycle phase. The advent of mass cytometry (MCM) provides tremendous potential for high throughput single cell analysis through direct measurements of elemental isotopes, and the development of a method to measure the cell cycle state by MCM further extends the utility of MCM. Here we describe a method that directly measures 5-iodo-2&#8242;-deoxyuridine (IdU), similar to 5-bromo-2&#180;-deoxyuridine (BrdU), in an MCM system. Use of this IdU-based MCM provides several advantages. First, IdU is rapidly incorporated into DNA during its synthesis, allowing reliable measurement of cells in the S-phase with incubations as short as 10-15 minutes. Second, IdU is measured without the need for secondary antibodies or the need for DNA degradation. Third, IdU staining can be easily combined with measurement of cyclin B1, phosphorylated retinoblastoma protein (pRb), and phosphorylated histone H3 (pHH3), which collectively provides clear delineation of the five cell cycle phases. Combination of these cell cycle markers with the high number of parameters possible with MCM allow combination with numerous other metrics.

Use of the Pyrimidine Analog, 5-Iodo-2&#8242;-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

This protocol adapts cell cycle measurements for use in a mass cytometry platform. With the multi-parameter capabilities of mass cytometry, direct measurement of iodine incorporation allows identification of cells in S-phase while intracellular cycling markers enable characterization of each cell cycle state in a range of experimental conditions.

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the ...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

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 ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) has been utilized to investigate replicative/S-phase progression, whereas antibodies against phospho-histone H3 have been utilized to mark mitotic nuclei and cells. A combination of both labels would enable the classification of G0/G1 (Gap phase), S (replicative), and M (mitotic) phases and serve as an important tool to evaluate the effects of mitotic gene knockdowns or null mutants on cell cycle progression. However, the reagents used to mark EdU-labelled cells are incompatible with several secondary antibody-fluorescent tags. This complicates immunostaining, where primary and tagged secondary antibodies are used to mark pH3-positive mitotic cells. This paper describes a step-by-step protocol for the dual-labeling of EdU and pH3 in Drosophila larval neural stem cells, a system utilized extensively to study mitotic factors. Additionally, a protocol is provided for image analysis and quantification to allocate labeled cells in 3 distinct categories, G0/G1, S, S&gt;G2/M (progression from S to G2/M), and M phases.

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

Cell cycle analysis with 5-ethynyl-2'-deoxyuridine (EdU) and phospho-histone H3 (pH3) labeling is a multi-step procedure that may require extensive optimization. Here, we present a detailed protocol that describes all steps for this procedure including image analysis and quantification to distinguish cells in different cell cycle phases.

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) ...

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...

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

Summary

Explore More Videos

Chapters in this video

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

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Use of the Pyrimidine Analog, 5-Iodo-2′-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae