Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 2FA, UK.
Mol Cell. 2012 Mar 9;45(5):696-704. doi: 10.1016/j.molcel.2012.01.007. Epub 2012 Feb 9.
The S phase checkpoint pathway preserves genome stability by protecting defective DNA replication forks, but the underlying mechanisms are still understood poorly. Previous work with budding yeast suggested that the checkpoint kinases Mec1 and Rad53 might prevent collapse of the replisome when nucleotide concentrations are limiting, thereby allowing the subsequent resumption of DNA synthesis. Here we describe a direct analysis of replisome stability in budding yeast cells lacking checkpoint kinases, together with a high-resolution view of replisome progression across the genome. Surprisingly, we find that the replisome is stably associated with DNA replication forks following replication stress in the absence of Mec1 or Rad53. A component of the replicative DNA helicase is phosphorylated within the replisome in a Mec1-dependent manner upon replication stress, and our data indicate that checkpoint kinases control replisome function rather than stability, as part of a multifaceted response that allows cells to survive defects in chromosome replication.
S 期检查点通路通过保护有缺陷的 DNA 复制叉来维持基因组稳定性，但其中的底层机制仍未得到充分理解。之前在芽殖酵母中的研究表明，检查点激酶 Mek1 和 Rad53 可能会在核苷酸浓度有限时防止复制体的崩溃，从而允许随后重新开始 DNA 合成。在这里，我们描述了对缺乏检查点激酶的芽殖酵母细胞中的复制体稳定性的直接分析，以及对基因组范围内复制体进展的高分辨率观察。令人惊讶的是，我们发现，在没有 Mek1 或 Rad53 的情况下，复制体在复制应激后仍能稳定地与 DNA 复制叉结合。在复制应激下，复制体中的一个复制性 DNA 解旋酶成分以 Mek1 依赖性的方式被磷酸化，并且我们的数据表明，检查点激酶控制复制体的功能而不是稳定性，这是细胞能够在染色体复制缺陷的情况下存活的多方面反应的一部分。
