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

Summary

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.

Abstract

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.

Introduction

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 phase^4,5,6. Consequently, these deregulations, associated or not with replication stress, result in chromosome breaks, rearrangements, and mis-segregation^4,5,6. 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.

Protocol

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.

1. 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.
   1. Inoculate S. cerevisiae cells in 10 mL of synthetic complete (SC) medium at a low cell concentration (5 × 10⁴ cells/mL) for an overnight culture at 30 ˚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 × 10⁷ cells/mL). Growing cells in rich medium (YPD) is not recommended, as EdU detection is not efficient.
   2. The following day, dilute the cells in 20 mL of fresh SC medium at the final concentration of 5 × 10⁵ cells/mL.
   3. Culture the cells at 30 ˚C in a shaking water bath with horizontal shaking at 120 rpm.
   4. Measure the cell concentration every hour until it reaches 1 × 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.
   5. Proceed in parallel to step 2 for EdU labeling when the cell concentration is about 2 × 10⁶-5 × 10⁶ cells/mL.
2. 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.
   1. Inoculate S. cerevisiae cells in 10 mL of SC medium at a low cell concentration (5 × 10⁴ cells/mL) for an overnight culture at 30 ˚C with orbital agitation at 130 rpm.
      NOTE: See the notes after step 1.1.1.
   2. The following day, dilute the cells in 20 mL of fresh SC medium at a final concentration of 2 × 10⁶-3 × 10⁶ cells/mL.
   3. Add 40 µL of 1 mg/mL α-factor diluted in water.
   4. Culture the cells at 30 ˚C with orbital agitation at 130 rpm for 1 h.
   5. Add again 40 µL of 1 mg/mL α-factor diluted in water.
   6. Culture the cells at 30 ˚C with orbital agitation at 130 rpm for 1 h.
   7. 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.
   8. Centrifuge for 3 min at 1,500 × g. Discard the supernatant with a vacuum pipette.
   9. Resuspend the cells in 20 mL of SC medium.
   10. Repeat steps 1.2.8-1.2.9 once.
       NOTE: The α-factor is washed away with these steps, and the cells are released in the cell cycle. Alternatively, the α-factor may be washed away by filtering the yeast cells with a 1.2 µm nitrocellulose filter using a funnel set on a side-arm flask connected to a vacuum pump.
   11. 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.
   12. Add 400 µL of 1 mg/mL α-factor diluted in water 30 min after the release.
       ​NOTE: This high dose of α-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.

2. EdU labeling

1. Transfer 1 mL of cell culture to a 2.0 mL microfuge tube containing 1 µL 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 µL of DMSO.
2. Incubate for 3-5 min at 30 ˚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.
3. Stop the reaction with the addition of 100 µL of 100% ethanol.
   1. Stop the reaction with the addition of 100 µL 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.
   2. 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 µL of 100% ethanol.

3. Cell fixation and permeabilization

1. Pellet the cells for 2 min at 10,000 × g in a microfuge. Remove the supernatant using a vacuum pipette.
2. Resuspend the cell pellet in 500 µL of 70% ethanol. Mix well by vortexing.
3. Leave for ≥1 h at RT at 20 tilts/min on a variable speed rocker to permeabilize the cells.
   ​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 ˚C overnight or for longer periods at −20 ˚C.
4. Pellet the cells for 2 min at 10,000 × g in a microcentrifuge. Discard the supernatant with a vacuum pipette.
5. Wash the cells 2x with 500 µL of 10% ethanol in PBS.
   ​NOTE: The washes are crucial to remove unincorporated EdU from the cells.

4. Click-it reaction

1. For cytometry analysis
   1. Pellet the cells for 2 min at 10,000 × g in a microfuge. Discard the supernatant with a vacuum pipette.
   2. Resuspend the pellet in 200 µL of PBS containing 0.1 mg/mL RNase A and 0.2 mg/mL proteinase K.
   3. Incubate for 1-2 h at 50 °C with occasional shaking (or overnight at 37 °C).
   4. Pellet the cells for 2 min at 10,000 × g in a microcentrifuge. Discard the supernatant with a vacuum pipette.
   5. Wash the cells with 500 µL of PBS.
   6. Pellet the cells for 2 min at 10,000 × g in a microfuge. Discard the supernatant with a vacuum pipette.
   7. Resuspend the cell pellet in 200 µL 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.
   8. Pellet the cells for 2 min at 10,000 × g in a microcentrifuge. Discard the supernatant with a vacuum pipette.
   9. Resuspend the pellet in 300 µL of PBS + 1% BSA.
   10. Distribute the cells between two tubes: 200 µL in a 1.5 mL microcentrifuge tube for the Click reaction and 100 µL in another 1.5 mL microcentrifuge tube for Sytox Green staining.
   11. Pellet the cells for 2 min at 10,000 × g in a microcentrifuge. Discard the supernatant with a vacuum pipette.
   12. 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.
       1. Resuspend the cell pellet in 100 µL of PBS.
       2. Transfer 10-30 µL (depending on the cell concentration) to a flow cytometer tube containing 300 µL of 50 mM Tris-HCl, pH 7.5, and 0.5 µM Sytox Green.
       3. Sonicate 2x, for 2 s each time, at an amplitude of 40-50.
       4. Leave in the dark until processing the samples on a flow cytometer.
          NOTE: The cells can be kept at this stage at 4 ˚C for a few days.
   13. For the Click reaction
       1. Prepare fresh Azide Dye buffer by mixing the reagents in the following order (quantity for one tube): 36 µL of PBS, 2 µL of 0.2 M CuSO₄, 0.2 µL of 2 mM Cy5 Azide, and 2 µL 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.
       2. Resuspend the cell pellet with 40 µL of freshly made Azide Dye mix. Incubate at RT in the dark for 60 min.
       3. Pellet the cells for 2 min at 10,000 × g in a microcentrifuge. Discard the supernatant with a vacuum pipette.
       4. Wash the cells 3x with 300 µL of 10% ethanol in PBS.
          NOTE: The washes are crucial to eliminate all the soluble EdU-Cy5 azide.
       5. Resuspend the cells in 100 µL of 50 µg/mL PI in PBS. Leave for 10 min in the dark.
       6. Transfer 10-30 µL of the cell suspension (depending on the cell concentration) to a flow cytometer tube containing 300 µL of 50 mM Tris-HCl, pH 7.5.
       7. Sonicate 2x, for 2 s each time, at an amplitude of 40-50.
       8. Leave in the dark until processing the samples on a cytometer.
          NOTE: The cells can be kept at this stage at 4 ˚C for a few days.
   14. 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.
2. For microscopy analysis
   1. Pellet the cells for 2 min at 10,000 × g in a microcentrifuge. Discard the supernatant with a vacuum pipette.
   2. Resuspend the pellet in 200 µL of PBS + 1% BSA. Incubate for 30 min at RT.
   3. Pellet the cells for 2 min at 10,000 × g in a microfuge. Discard the supernatant with a vacuum pipette.
   4. Prepare fresh Azide Dye buffer by mixing the reagents in the following order (quantity for one tube): 36 µL of PBS, 2 µL of 0.2 M CuSO₄, 0.2 µL of 2 mM Dy-530 azide, 2 µL 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.
   5. Resuspend the pellet with 40 µL of freshly made Azide Dye buffer. Incubate at RT in the dark for 60 min.
   6. Pellet the cells for 2 min at 10,000 × g in a microfuge. Discard the supernatant with a vacuum pipette.
   7. Wash the cells 2x with 300 µL of 10% ethanol in PBS.
      NOTE: The washes are crucial to eliminate all soluble Dy-530 azide.
   8. Pellet the cells for 2 min at 10,000 × g in a microcentrifuge. Discard the supernatant with a vacuum pipette.
   9. Resuspend the cells in 100 µL of 0.5 µg/mL 4',6-diamidino-2-phenylindole (DAPI) in PBS. Leave for 30 min in the dark at room temperature.
   10. Pellet the cells for 2 min at 10,000 × g in a microcentrifuge. Discard the supernatant with a vacuum pipette.
   11. Wash with 300 µL of PBS to remove excess DAPI.
   12. Pellet the cells for 2 min at 10,000 × g in a microfuge. Discard the supernatant with a vacuum pipette.
   13. Resuspend the pellet with 10-50 µL of PBS depending on the cell concentration.
   14. Sonicate 2x, for 2 s each time, at an amplitude of 40-50.
       NOTE: The cells can be kept at this stage at 4 ˚C for a few days.
   15. Pipette 1.7 µL of the cells onto a glass microscope slide and cover with a clean coverslip.
   16. Immediately observe under a fluorescence microscope with DAPI and TexasRed or Cy3 filters.

Results

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 ± 13 min at 25 ˚C (Table 2). When the cells were in the ex...

Discussion

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 problem^15,16. EdU is more versatile than BrdU as its detection with smal...

Materials

Name	Company	Catalog Number	Comments
α-factor	Genescript	RP01002
Bovine Serum Albumin (BSA)	Euromedex	04-100--812-E
Copper sulfate	Sigma	C1297
DAPI	Sigma	D9542
Di-sulfo-Cyanine5 azide (Cy5 azide)	Interchim	FP-JV6320	Alternative to Alexa647-Azide
Dy-530 azide	Dyomics	530-10
EdU (5-ethynyl-2’-deoxyuridine)	Carbosynth	NE08701
Ethanol absolute	Carlo Erba reagents	P013A10D16	or equivalent
L- ascorbic acid	Sigma	A4544
Propidium iodide	Sigma	P4864
Proteinase K	Euromedex	EU0090
Rnase	SIGMA	R5000
Sytox Green	Invitrogen	S-7020
Equipment
Cell counter	OLS	CASY
Flow cytometer	Agilent	NovoSampler Pro
Shaking incubator	Infors	444-4230	or equivalent
Shaking water bath	Julabo	SW22	or equivalent
Sonicator	Sonics	Vibra cell
Wide-field microscopy	Leica	THUNDER Imager	or equivalent