Department of Molecular and Cellular Phamacology, University of Miami School of Medicine PO Box 016189, Miami, FL 33140, USA.
Cell Div. 2010 Aug 27;5:20. doi: 10.1186/1747-1028-5-20.
In fission yeast the intra-S phase and DNA damage checkpoints are activated in response to inhibition of DNA replication or DNA damage, respectively. The intra-S phase checkpoint responds to stalled replication forks leading to the activation of the Cds1 kinase that both delays cell cycle progression and stabilizes DNA replication forks. The DNA damage checkpoint, that operates during the G2 phase of the cell cycle delays mitotic progression through activation of the checkpoint kinase, Chk1. Delay of the cell cycle is believed to be essential to allow time for either replication restart (in S phase) or DNA damage repair (in G2). Previously, our laboratory showed that fission yeast cells deleted for the N-terminal half of DNA polymerase ε (Cdc20) are delayed in S phase, but surprisingly require Chk1 rather than Cds1 to maintain cell viability. Several additional DNA replication mutants were then tested for their dependency on Chk1 or Cds1 when grown under semi-permissive temperatures. We discovered that mutants defective in DNA replication initiation are sensitive only to loss of Chk1, whilst mutations that inhibit DNA replication elongation are sensitive to loss of both Cds1 and Chk1. To confirm that the Chk1-sensitive, Cds1-insensitive phenotype (rid phenotype) is specific to mutants defective in DNA replication initiation, we completed a genetic screen for cell cycle mutants that require Chk1, but not Cds1 to maintain cell viability when grown at semi-permissive temperatures. Our screen identified two mutants, rid1-1 and rid2-1, that are defective in Orc1 and Mcm4, respectively. Both mutants show defects in DNA replication initiation consistent with our hypothesis that the rid phenotype is replication initiation specific. In the case of Mcm4, the mutation has been mapped to a highly conserved region of the protein that appears to be required for DNA replication initiation, but not elongation. Therefore, we conclude that the cellular response to inhibition of DNA replication initiation is distinct from blocking DNA replication elongation, and this difference can be exploited to identify mutants specifically defective in DNA replication initiation.
在裂殖酵母中，内 S 期和 DNA 损伤检查点分别响应于 DNA 复制的抑制或 DNA 损伤而被激活。内 S 期检查点响应于停滞的复制叉，导致 Cds1 激酶的激活，该激酶既延迟细胞周期进程，又稳定 DNA 复制叉。DNA 损伤检查点在细胞周期的 G2 期起作用，通过激活检查点激酶 Chk1 延迟有丝分裂进程。细胞周期的延迟被认为是必需的，以便为复制启动（在 S 期）或 DNA 损伤修复（在 G2 期）留出时间。此前，我们实验室表明，缺失 DNA 聚合酶 ε（Cdc20）N 端一半的裂殖酵母细胞在 S 期被延迟，但令人惊讶的是，为了维持细胞活力，它们需要 Chk1 而不是 Cds1。然后，我们测试了其他几个 DNA 复制突变体在半许可温度下生长时对 Chk1 或 Cds1 的依赖性。我们发现，复制起始缺陷的突变体仅对 Chk1 的缺失敏感，而抑制 DNA 复制延伸的突变体对 Cds1 和 Chk1 的缺失都敏感。为了确认 Chk1 敏感、Cds1 不敏感表型（rid 表型）特异性针对复制起始缺陷的突变体，我们完成了一个针对需要 Chk1 但不需要 Cds1 才能在半许可温度下维持细胞活力的细胞周期突变体的遗传筛选。我们的筛选确定了两个突变体 rid1-1 和 rid2-1，它们分别在 Orc1 和 Mcm4 中缺陷。这两个突变体都显示出与我们的假设一致的 DNA 复制起始缺陷，即 rid 表型是复制起始特异性的。在 Mcm4 的情况下，该突变已被定位到该蛋白的高度保守区域，该区域似乎对于 DNA 复制起始是必需的，但不是延伸所必需的。因此，我们得出结论，细胞对 DNA 复制起始抑制的反应与阻断 DNA 复制延伸不同，并且可以利用这种差异来鉴定专门在 DNA 复制起始中缺陷的突变体。
