Yabuuchi Hayato, Yamada Yoshiki, Uchida Tomonori, Sunathvanichkul Tul, Nakagawa Takuro, Masukata Hisao
Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
EMBO J. 2006 Oct 4;25(19):4663-74. doi: 10.1038/sj.emboj.7601347. Epub 2006 Sep 21.
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.
真核生物中染色体DNA复制的起始通过复制因子在复制起点的组装受到严格调控。在此，我们利用温度敏感型裂殖酵母突变体研究了起始复合物组装对特定因子的依赖性。psf3-1突变体是一种GINS组分突变体，在限制温度下因DNA未复制而停滞，且DNA含量逐渐增加，表明DNA复制存在缺陷。下拉实验表明该突变损害了GINS复合物的形成。染色质免疫沉淀分析表明，GINS的完整性是Psf2、Cut5和Cdc45在起点加载所必需的，但不是Sld3。相反，Psf2在起点的加载依赖于Sld3和Cut5，而不依赖于Cdc45。这些结果表明，Sld3在起始复合物组装中作用于最上游，其次是GINS和Cut5，然后是Cdc45。与这一结论一致，Sld3的加载需要Cdc7-Dbf4激酶（DDK）而不是细胞周期蛋白依赖性激酶（CDK），而其他因子的募集则依赖于这两种激酶。这些结果表明，DDK和CDK在裂殖酵母中调控复制起点激活的不同步骤。
