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

Juan de Dios Barba Tena; Etienne Schwob; Nicolas Talarek

doi:10.3791/64490

Determinazione della durata della fase S mediante incorporazione di 5-etinil-2'-deossiuridina in ...

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

Determinazione della durata della fase S mediante incorporazione di 5-etinil-2'-deossiuridina in Saccharomyces cerevisiae

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08:40 min

October 21, 2022

DOI: 10.3791/64490-v

Juan de Dios Barba Tena¹, Etienne Schwob¹, Nicolas Talarek¹

¹Institut de Génétique Moléculaire de Montpellier,Université de Montpellier, CNRS

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Overview

This study describes a precise method to determine S-phase duration in the model organism Saccharomyces cerevisiae. The protocol utilizes EdU, a thymidine analog, combined with Click chemistry to identify DNA synthesis and potentially overlooked replication defects in yeast mutants.

Key Study Components

Research Area

Cell cycle regulation
DNA replication
Mutant analysis

Background

Budding yeast as a model organism
Significance of S-phase duration
Previous limitations of classical protocols

Methods Used

EdU incorporation and Click chemistry
Saccharomyces cerevisiae
Microscopy and flow cytometry

Main Results

Successfully measured S-phase duration of approximately 25 minutes
Identified 29% of cells in the S-phase during analysis
Detected mild synthesis defects not apparent in classical methods

Conclusions

The protocol provides a reliable approach for studying S-phase duration
Implications for further research on cell cycle dynamics in yeast

Frequently Asked Questions

What is EdU?

EdU is a thymidine analog used to label cells undergoing DNA synthesis.

Why use budding yeast for this study?

Saccharomyces cerevisiae is a well-established model for studying eukaryotic cell biology.

How does Click chemistry work in this protocol?

Click chemistry is used to label EdU incorporated in DNA, allowing detection via microscopy and flow cytometry.

What are the advantages of this method?

The method is quick, easy, reproducible, and sensitive enough to detect slight replication defects.

What does S-phase duration indicate?

S-phase duration is critical for understanding DNA replication timing and its regulation during the cell cycle.

Can this method be applied to other organisms?

While this method is optimized for yeast, it may be adapted for other cell types with appropriate adjustments.

What future applications may arise from this research?

Insights from this study may facilitate research in cancer biology, genetics, and cell cycle regulation.

Descriviamo due protocolli complementari per determinare con precisione la durata della fase S in S. cerevisiae utilizzando EdU, un analogo della timidina, che viene incorporato in vivo e rilevato utilizzando la chimica Click mediante microscopia e citometria a flusso. Permette una facile caratterizzazione della durata della replicazione del DNA e dei difetti di replicazione trascurati nei mutanti.

Questo protocollo consente una determinazione precisa della durata della fase S in cellule di lievito in erba sincronizzate. È un metodo rapido, facile e riproducibile. Inoltre, è abbastanza sensibile da rilevare lievi difetti di sintesi non rilevati dai protocolli classici.

Questo metodo sarà utile per molti ricercatori che lavorano sulla regolazione del ciclo cellulare nel lievito in erba. A dimostrare questo protocollo saranno Nicolas Talarek, un ricercatore senior, e Juan De Dios Barba Tena, uno studente di dottorato in laboratorio. Inoculare le cellule di Saccharomyces cerevisiae in 10 millilitri di terreno SC a bassa concentrazione cellulare per una coltura notturna a 30 gradi Celsius con agitazione orbitale a 130 rotazioni al minuto.

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