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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.
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.
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....
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.
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.......
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.......
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.
....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 |
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