Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins
Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins
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DOI：
10.1038/sj.emboj.7601347
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发表时间：
2006-10-04
期刊：
影响因子：
11.4
通讯作者：
Masukata, Hisao
中科院分区：
文献类型：
--
作者：
Yabuuchi, Hayato;Yamada, Yoshiki;Masukata, Hisao
真核生物中染色体 DNA 复制的启动受到复制起点处复制因子组装的严格调控。在这里，我们使用温度敏感的裂殖酵母突变体研究了起始复合物的组装对特定因素的依赖性。 psf3-1突变体是GINS组分突变体，在限制温度下与未复制的DNA一起停滞，并且DNA含量逐渐增加，表明DNA复制存在缺陷。正如 Pull-down 实验所示，突变损害了 GINS 复合物的形成。染色质免疫沉淀分析表明，Psf2、Cut5 和 Cdc45 的起始加载需要 GINS 完整性，但 Sld3 则不需要。相比之下，将 Psf2 加载到起点上取决于 Sld3 和 Cut5，而不取决于 Cdc45。这些结果表明，Sld3 在起始复合物组装中发挥最上游的作用，其次是 GINS 和 Cut5，然后是 Cdc45。与此结论一致，Sld3 加载需要 Cdc7-Dbf4 激酶 (DDK) 而不是细胞周期蛋白依赖性激酶 (CDK)，而其他因子的招募取决于这两种激酶。这些结果表明，DDK 和 CDK 调节裂殖酵母中复制起点激活的不同步骤。
Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.