Nagata Mariko, Ishino Sonoko, Yamagami Takeshi, Simons Jan-Robert, Kanai Tamotsu, Atomi Haruyuki, Ishino Yoshizumi
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan.
Sci Rep. 2017 Dec 5;7(1):16949. doi: 10.1038/s41598-017-17306-0.
RecJ was originally identified in Escherichia coli and plays an important role in the DNA repair and recombination pathways. Thermococcus kodakarensis, a hyperthermophilic archaeon, has two RecJ-like nucleases. These proteins are designated as GAN (GINS-associated nuclease) and HAN (Hef-associated nuclease), based on the protein they interact with. GAN is probably a counterpart of Cdc45 in the eukaryotic CMG replicative helicase complex. HAN is considered mainly to function with Hef for restoration of the stalled replication fork. In this study, we characterized HAN to clarify its functions in Thermococcus cells. HAN showed single-strand specific 3' to 5' exonuclease activity, which was stimulated in the presence of Hef. A gene disruption analysis revealed that HAN was non-essential for viability, but the ΔganΔhan double mutant did not grow under optimal conditions at 85 °C. This deficiency was not fully recovered by introducing the mutant han gene, encoding the nuclease-deficient HAN protein, back into the genome. These results suggest that the unstable replicative helicase complex without GAN performs ineffective fork progression, and thus the stalled fork repair system including HAN becomes more important. The nuclease activity of HAN is required for the function of this protein in T. kodakarensis.
RecJ最初是在大肠杆菌中发现的，在DNA修复和重组途径中发挥重要作用。嗜热栖热菌是一种嗜热古菌，有两种RecJ样核酸酶。根据它们相互作用的蛋白质，这些蛋白质被命名为GAN（GINS相关核酸酶）和HAN（Hef相关核酸酶）。GAN可能是真核生物CMG复制解旋酶复合物中Cdc45的对应物。HAN主要被认为与Hef一起发挥作用，以恢复停滞的复制叉。在本研究中，我们对HAN进行了表征，以阐明其在嗜热栖热菌细胞中的功能。HAN表现出单链特异性3'至5'核酸外切酶活性，在Hef存在的情况下这种活性会增强。基因敲除分析表明，HAN对生存力不是必需的，但ΔganΔhan双突变体在85°C的最佳条件下不能生长。通过将编码核酸酶缺陷型HAN蛋白的突变han基因重新引入基因组，这种缺陷并未完全恢复。这些结果表明，没有GAN的不稳定复制解旋酶复合物进行的叉状进展无效，因此包括HAN在内的停滞叉修复系统变得更加重要。HAN的核酸酶活性是该蛋白在嗜热栖热菌中发挥功能所必需的。
