Hustedt Nicole, Shimada Kenji
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058, Basel, Switzerland.
Methods Mol Biol. 2014;1170:321-41. doi: 10.1007/978-1-4939-0888-2_16.
Checkpoints are conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. Cells also possess a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication fork progression. In the budding yeast Saccharomyces cerevisiae, the DNA replication checkpoint controls the timing of S-phase events such as origin firing and spindle elongation. This checkpoint also upregulates dNTP pools and maintains the replication fork structure in order to resume DNA replication after replication block. Many replication checkpoint factors have been found to be tumor suppressors, highlighting the importance of this checkpoint pathway in human health. Here we describe a series of protocols to analyze the DNA replication checkpoint in S. cerevisiae.
关卡是保守机制，当细胞无法正常完成上一事件时，可阻止其进入细胞周期的下一阶段。细胞还拥有一种称为DNA复制关卡的监测机制，它由一个保守的激酶级联组成，该激酶级联由阻碍或减缓复制叉进展的损伤引发。在芽殖酵母酿酒酵母中，DNA复制关卡控制S期事件的时间，如复制起点激发和纺锤体伸长。该关卡还上调dNTP库并维持复制叉结构，以便在复制受阻后恢复DNA复制。已发现许多复制关卡因子是肿瘤抑制因子，这突出了该关卡途径在人类健康中的重要性。在这里，我们描述了一系列用于分析酿酒酵母中DNA复制关卡的实验方案。
