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

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Biology

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

使用酿酒酵母中的5-乙炔基-2'-脱氧尿苷掺入测定S相持续时间

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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.

我们描述了两种互补的方案，以使用EdU（一种胸苷类似物）准确确定 酿酒酵母 的S期持续时间，该EdU在体内掺入并通过显微镜和流式细胞术使用Click化学进行检测。它可以轻松表征突变体中DNA复制的持续时间和被忽视的复制缺陷。

该协议允许精确确定同步出芽酵母细胞中的S相持续时间。这是一种快速、简单且可重复的方法。此外，它足够灵敏，可以检测经典方案无法检测到的轻度合成缺陷。

这种方法对于许多研究出芽酵母细胞周期调节的研究人员很有用。展示该协议的将是高级研究员Nicolas Talarek和实验室的博士生Juan De Dios Barba Tena。在10毫升SC培养基中以低细胞浓度接种酿酒酵母细胞，以30摄氏度的速度进行过夜培养，以每分钟130转的速度进行轨道搅拌。

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