利用裂殖酵母粟酒裂殖酵母研究S期DNA损伤检查点。
Studying S-phase DNA damage checkpoints using the fission yeast Schizosaccharomyces pombe.
作者信息
Willis Nicholas, Rhind Nicholas
机构信息
Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
出版信息
Methods Mol Biol. 2011;782:13-21. doi: 10.1007/978-1-61779-273-1_2.
Slowing of replication in response to DNA damage is a universal response to DNA damage during S-phase. Originally discovered to be defective in checkpoint mutant cells in metazoans, this S-phase DNA damage checkpoint response has been extensively studied in yeast. Unlike other checkpoints that completely arrest cell cycle, the S-phase DNA damage checkpoint slows but does not completely halt replication in response to DNA damage. An analysis of mutants defective in the slowing response requires a sensitive assay to measure this quantitative effect. The use of centrifugal elutriation to synchronize cells and improved techniques in preparing cells for flow cytometry allow for more sensitive and accurate measurement of cells' ability to slow replication in the presence of DNA damage. This chapter describes the use of transient cdc10-M17 temperature sensitive allele arrest and release combined with centrifugal elutriation to synchronize cells in G1. The S-phase progression of these cells is then assayed by flow cytometry of isolated nuclei, which allows sensitive determination of replication kinetics.
响应DNA损伤时复制减缓是S期对DNA损伤的一种普遍反应。最初发现后生动物的检查点突变细胞中存在缺陷，这种S期DNA损伤检查点反应已在酵母中得到广泛研究。与其他完全阻滞细胞周期的检查点不同，S期DNA损伤检查点会减缓但不会完全停止对DNA损伤的复制反应。对减缓反应缺陷的突变体进行分析需要一种灵敏的检测方法来测量这种定量效应。使用离心淘洗来同步细胞以及改进用于流式细胞术的细胞制备技术，能够更灵敏、准确地测量细胞在DNA损伤存在时减缓复制的能力。本章描述了使用瞬时cdc10 - M17温度敏感等位基因阻滞和释放并结合离心淘洗来使细胞在G1期同步化。然后通过对分离细胞核的流式细胞术来检测这些细胞的S期进程，这可以灵敏地确定复制动力学。
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