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, duplica....

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 ....

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