Nagata Mariko, Ishino Sonoko, Yamagami Takeshi, Ogino Hiromi, Simons Jan-Robert, Kanai Tamotsu, Atomi Haruyuki, Ishino Yoshizumi
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Fukuoka 812-8581, Japan.
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Kyoto 615-8510, Japan.
Nucleic Acids Res. 2017 Oct 13;45(18):10693-10705. doi: 10.1093/nar/gkx740.
The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells.
古菌微小染色体维持蛋白（MCM）具有DNA解旋酶活性，在几种古菌中，该活性受GINS刺激。在真核生物复制性解旋酶复合体中，Cdc45与MCM和GINS形成复合体，命名为CMG（Cdc45-MCM-GINS）。Cdc45与细菌RecJ具有序列相似性。来自嗜热栖热菌的一种Cdc45/RecJ样蛋白表现出细菌RecJ样核酸外切酶活性，在体外该活性受GINS刺激。因此，这种古菌Cdc45/RecJ被命名为GAN，即GINS相关核酸酶。在本研究中，我们在嗜热栖热菌细胞中鉴定出了CMG样复合体。GAN·GINS复合体刺激MCM解旋酶，但在体外MCM不影响GAN的核酸酶活性。基因敲除分析表明，GAN对其生存力并非必需，但Δgan突变体在93°C下无法生长。此外，与亲本细胞相比，Δgan突变体在85°C的最佳条件下生长明显迟缓。通过将编码核酸酶缺陷型GAN蛋白的gan基因重新导入基因组，这些缺陷得以恢复。这些结果表明，不含GAN的复制性解旋酶复合体可能会变得不稳定，且在复制叉推进过程中效率低下。GAN的核酸酶活性与Δgan突变体细胞的生长缺陷无关。
