Sugimoto K, Shimomura T, Hashimoto K, Araki H, Sugino A, Matsumoto K
Department of Molecular Biology, Faculty of Science, Nagoya University, Japan.
Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7048-52. doi: 10.1073/pnas.93.14.7048.
The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.
DNA合成的抑制通过S期检查点的作用阻止有丝分裂的进入。在酿酒酵母中，一种必需的蛋白激酶Spk1/Mec2/Rad53/Sad1控制S期与有丝分裂的偶联。为了鉴定与Spk1发生遗传相互作用的基因，我们分离出了一个温度敏感突变体rfc5-1，它可以被SPK1的过表达所抑制。RFC5基因编码复制因子C复合物的一个小亚基。在限制温度下，rfc5-1突变体细胞进入有丝分裂时染色体分离不均或断裂，导致活力丧失。因此，DNA复制缺陷的rfc5突变体在S期检查点也有缺陷。编码增殖细胞核抗原的POL30的过表达抑制了rfc5突变体的复制缺陷，但没有抑制其检查点缺陷。综上所述，这些结果表明复制因子C在感知DNA复制状态并将信号传递给检查点机制方面具有直接作用。
