Institute of Human Genetics, Centre National de Recherche Scientifique, Unité Propre de Recherche 1142, Montpellier, France.
Nat Struct Mol Biol. 2010 Nov;17(11):1391-7. doi: 10.1038/nsmb.1932. Epub 2010 Oct 24.
Maintenance of genome integrity relies on surveillance mechanisms that detect and signal arrested replication forks. Although evidence from budding yeast indicates that the DNA replication checkpoint (DRC) is primarily activated by single-stranded DNA (ssDNA), studies in higher eukaryotes have implicated primer ends in this process. To identify factors that signal primed ssDNA in Saccharomyces cerevisiae, we have screened a collection of checkpoint mutants for their ability to activate the DRC, using the repression of late origins as readout for checkpoint activity. This quantitative analysis reveals that neither RFC(Rad24) and the 9-1-1 clamp nor the alternative clamp loader RFC(Elg1) is required to signal paused forks. In contrast, we found that RFC(Ctf18) is essential for the Mrc1-dependent activation of Rad53 and for the maintenance of paused forks. These data identify RFC(Ctf18) as a key DRC mediator, potentially bridging Mrc1 and primed ssDNA to signal paused forks.
基因组完整性的维持依赖于能够检测和发出信号的监控机制，以阻止复制叉的停滞。尽管来自 budding yeast 的证据表明，DNA 复制检查点（DRC）主要通过单链 DNA（ssDNA）激活，但在高等真核生物中的研究表明引物末端也参与了这个过程。为了鉴定在 Saccharomyces cerevisiae 中发出引物 ssDNA 的信号因子，我们使用迟滞起始点的抑制作为检查点活性的读数，筛选了一组检查点突变体，以检测它们激活 DRC 的能力。这种定量分析表明，RFC（Rad24）和 9-1-1 夹以及替代的夹加载器 RFC（Elg1）都不需要发出停滞的叉信号。相比之下，我们发现 RFC（Ctf18）对于 Mrc1 依赖性的 Rad53 激活和停滞叉的维持是必需的。这些数据表明，RFC（Ctf18）是 DRC 中介的关键，可能将 Mrc1 和引物 ssDNA 桥接起来，以发出停滞的叉信号。
