Institut de Génétique Moléculaire de Montpellier, Université de Montpellier, CNRS.
Institut de Génétique Moléculaire de Montpellier, Université de Montpellier, CNRS;
J Vis Exp. 2022 Oct 21(188). doi: 10.3791/64490.
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.
真核生物 DNA 复制是一个高度调控的过程，可确保在染色体分离之前正确复制细胞的遗传蓝图。由于 DNA 合成缺陷是染色体重排的基础，因此监测 DNA 复制对于理解基因组不稳定性的基础至关重要。酿酒酵母是研究细胞周期调控的经典模型，但关键的 DNA 复制参数，如 S 期细胞的比例或 S 期持续时间，仍然难以确定。该方案使用短而无毒的 5-乙炔基-2'-脱氧尿苷（EdU）脉冲处理工程化的 TK-hENT1 酵母细胞，然后通过点击反应进行检测，从而通过显微镜和流式细胞术以高时空分辨率在单细胞和群体水平上对 DNA 复制进行可视化和定量。该方法可以鉴定酵母突变体中 S 期和细胞周期进程中以前被忽视的缺陷，从而可以鉴定确保基因组稳定性所必需的新参与者。
