Su Ning Yuan, Flick Karin, Kaiser Peter
University of California, Irvine, Department of Biological Chemistry, College of Medicine, 240D Med Sci I, Irvine, CA 92697-1700, USA.
Mol Cell Biol. 2005 May;25(10):3875-85. doi: 10.1128/MCB.25.10.3875-3885.2005.
The Saccharomyces cerevisiae ubiquitin ligase SCF(Met30) is essential for cell cycle progression. To identify and characterize SCF(Met30)-dependent cell cycle steps, we used temperature-sensitive met30 mutants in cell cycle synchrony experiments. These experiments revealed a requirement for Met30 during both G(1)/S transition and M phase, while progression through S phase was unaffected by loss of Met30 function. Expression of the G(1)-specific transcripts CLN1, CLN2, and CLB5 was very low in met30 mutants, whereas expression of CLN3 was unaffected. However, overexpression of Cln2 could not overcome the G(1) arrest. Interestingly, overexpression of Clb5 could induce DNA replication in met30 mutants, albeit very inefficiently. Increased levels of Clb5 could not, however, suppress the cell proliferation defect of met30 mutants. Consistent with the DNA replication defects, chromatin immunoprecipitation experiments revealed significantly lower levels of the replication factors Mcm4, Mcm7, and Cdc45 at replication origins in met30 mutants than in wild-type cells. These data suggest that Met30 regulates several aspects of the cell cycle, including G(1)-specific transcription, initiation of DNA replication, and progression through M phase.
酿酒酵母泛素连接酶SCF(Met30)对细胞周期进程至关重要。为了鉴定和表征依赖SCF(Met30)的细胞周期步骤，我们在细胞周期同步实验中使用了温度敏感型met30突变体。这些实验揭示了在G(1)/S转换和M期期间对Met30的需求，而Met30功能丧失并不影响S期进程。met30突变体中G(1)特异性转录本CLN1、CLN2和CLB5的表达非常低，而CLN3的表达不受影响。然而，Cln2的过表达并不能克服G(1)期阻滞。有趣的是，Clb5的过表达可以在met30突变体中诱导DNA复制，尽管效率非常低。然而，Clb5水平的增加并不能抑制met30突变体的细胞增殖缺陷。与DNA复制缺陷一致，染色质免疫沉淀实验显示，met30突变体中复制起始点处的复制因子Mcm4、Mcm7和Cdc45的水平明显低于野生型细胞。这些数据表明，Met30调节细胞周期的多个方面，包括G(1)特异性转录、DNA复制起始和M期进程。
