Uchiyama M, Griffiths D, Arai K, Masai H
Department of Molecular and Developmental Biology, Institute for Medical Science, The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan.
J Biol Chem. 2001 Jul 13;276(28):26189-96. doi: 10.1074/jbc.M100007200. Epub 2001 May 8.
Assembly of replication complexes at the replication origins is strictly regulated. Cdc45p is known to be a part of the active replication complexes. In Xenopus egg extracts, Cdc45p was shown to be required for loading of DNA polymerase alpha onto chromatin. The fission yeast cdc45 homologue was identified as a suppressor for nda4 and named sna41. Nevertheless, it is not known how Cdc45p facilitates loading of DNA polymerase alpha onto chromatin, particularly to prereplicative complexes. To gain novel insight into the function of this protein in fission yeast, we characterized the fission yeast Cdc45 homologue, Sna41p. We have constructed C-terminally epitope-tagged Sna41p and Pol alpha p and replaced the endogenous genes with the corresponding tagged genes. Analyses of protein-protein interactions in vivo by the use of these tagged strains revealed the following: Sna41p interacts with Pol alpha p throughout the cell cycle, whereas it interacts with Mis5p/Mcm6p in the chromatin fractions at the G(1)-S boundary through S phase. In an initiation-defective sna41 mutant, sna41(goa1), interaction of Pol alpha p with Mis5p is not observed, although Pol alpha p loading onto the chromatin that occurs before G(1) START is not affected. These results show that fission yeast Sna41p facilitates the loading of Pol alpha p onto minichromosome maintenance proteins. Our results are consistent with a model in which loading of Pol alpha p onto replication origins occurs through two steps, namely, loading onto chromatin at preSTART and association with prereplicative complexes at G(1)-S through Sna41p, which interacts with minichromosome maintenance proteins in a cell cycle-dependent manner.
复制起始点处复制复合物的组装受到严格调控。已知Cdc45p是活性复制复合物的一部分。在非洲爪蟾卵提取物中，已表明Cdc45p是将DNA聚合酶α加载到染色质上所必需的。裂殖酵母的Cdc45同源物被鉴定为nda4的抑制因子，并命名为sna41。然而，尚不清楚Cdc45p如何促进DNA聚合酶α加载到染色质上，特别是加载到复制前复合物上。为了深入了解该蛋白在裂殖酵母中的功能，我们对裂殖酵母的Cdc45同源物Sna41p进行了表征。我们构建了C末端带有表位标签的Sna41p和Pol α p，并将内源性基因替换为相应的标签基因。利用这些标签菌株对体内蛋白质-蛋白质相互作用进行分析，结果如下：Sna41p在整个细胞周期中都与Pol α p相互作用，而在G(1)-S边界到S期的染色质组分中，它与Mis5p/Mcm6p相互作用。在起始缺陷的sna41突变体sna41(goa1)中，虽然在G(1) START之前发生的DNA聚合酶α加载到染色质上不受影响，但未观察到Pol α p与Mis5p的相互作用。这些结果表明，裂殖酵母Sna41p促进了Pol α p加载到微小染色体维持蛋白上。我们的结果与一个模型一致，即Pol α p加载到复制起始点是通过两个步骤进行的，即在preSTART时加载到染色质上，以及在G(1)-S期通过与微小染色体维持蛋白以细胞周期依赖性方式相互作用的Sna41p与复制前复合物结合。
