Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan.
Genes Cells. 2011 Dec;16(12):1176-89. doi: 10.1111/j.1365-2443.2011.01562.x. Epub 2011 Nov 17.
In eukaryotes, the replicative DNA helicase 'core' is the minichromosome maintenance (Mcm) complex (MCM), forming a heterohexameric complex consisting of six subunits (Mcm2-7). Recent studies showed that the CMG (Cdc45-MCM-GINS) complex is the actual helicase body in the replication fork progression complex. In Archaea, Thermococcus kodakarensis harbors three genes encoding the Mcm homologs on its genome, contrary to most archaea, which have only one homolog. It is thus, of high interest, whether and how these three Mcms share their functions in DNA metabolism in this hyperthermophile. Here, we report the biochemical properties of two of these proteins, TkoMcm1 and TkoMcm3. In addition, their physical and functional interactions with GINS, possibly an essential factor for the initiation and elongation process of DNA replication, are presented through in vitro ATPase and helicase assays, and an in vivo immunoprecipitation assay. Gene disruption and product quantification analyses suggested that TkoMcm3 is essential for cell growth and plays a key role as the main DNA helicase in DNA replication, whereas TkoMcm1 also shares some function in the cells.
在真核生物中，复制 DNA 解旋酶“核心”是微型染色体维持（Mcm）复合物（MCM），形成由六个亚基（Mcm2-7）组成的异六聚体复合物。最近的研究表明，CMG（Cdc45-MCM-GINS）复合物是复制叉推进复合物中的实际解旋酶体。在古菌中，与大多数古菌只有一个同源物不同，水生栖热菌含有三个编码 Mcm 同源物的基因。因此，这些三个 Mcm 如何在这种嗜热古菌的 DNA 代谢中共享其功能是非常有趣的。在这里，我们报告了其中两种蛋白，TkoMcm1 和 TkoMcm3 的生化特性。此外，通过体外 ATPase 和解旋酶测定以及体内免疫沉淀测定，还展示了它们与 GINS（可能是 DNA 复制起始和延伸过程的必需因素）的物理和功能相互作用。基因敲除和产物定量分析表明，TkoMcm3 对细胞生长是必需的，并且作为 DNA 复制的主要 DNA 解旋酶发挥关键作用，而 TkoMcm1 在细胞中也具有某些功能。
