平成26年度 医学研セミナー

Molecular analyses of replicative helicases in thermophilic archaea

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演者 石野 良純 教授(九州大学大学院農学研究院 生命機能科学部門 生物機能分子化学講座)
会場 東京都医学総合研究所 2BC会議室
日時 平成26年11月21日(金)18:00
世話人 正井 久雄 参事研究員(ゲノム動態プロジェクト)
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お問い合わせ 研究推進課 普及広報係


Regulatory mechanism for the accurate DNA replication is very important for maintenance of genome stability in the cells. In addition to this fundamental interest in molecular biology, hyperthermophiles are attractive target for the research on DNA replication to understand the living activity at high temperature. I will focus here on the molecules of replicative helicase core in Archaea. The MCM interacts with Cdc45 and GINS, to form a ternary assembly referred to as the “CMG complex”, that is believed to be the functional helicase in eukaryotic cells. In contrast to the eukaryotic MCM heterocomplex (Mcm2-7), the archaeal MCMs, consist of a homohexamer or homo double hexamer, having distinct DNA helicase activity by themselves in vitro.

The eukaryotic GINS complex consists of four different proteins, Sld5, Psf1, Psf2, and Psf3. The amino acid sequences of the four subunits in the GINS complex share some conservation, suggesting that they are ancestral paralogs. However, most of the archaeal genomes have only one gene encoding this family protein, and more interestingly, some archaea have two genes with sequences similar to Psf2 and Psf3, and Sld5 and Psf1, respectively referred to as Gins23 and Gins51. The GINS complex of tetrameric structure made of two proteins, Gins23 and Gins15, with a 2:2 molar ratio from S. solfataricus, P. furiosus, and T. kodakarensis have been characterized.

Thermoplasma acidophilum has a single Gins homolog, Gins51, and it forms a stable homotetramer (TaGINS). Furthermore, the helicase activity of TaMCM was clearly stimulated by TaGINS, as the case of P. furiosus, and T. kodakarensis. These results suggested that the MCM-GINS complex is a common part of the replicative helicase in Archaea. In addition, TaCdc6-2, one of the two Cdc6/Orc1 homologs in T. acidophilum, also stimulates the helicase activities of TaMCM in vitro, and it is a unique feature in archaea that the stimulation of the TaMCM helicase by TaGINS was enhanced by the addition of TaCdc6-2. Activation of the replicative helicase by a mechanism including a Cdc6-like protein suggests the divergent evolution after the division into Archaea and Eukarya.

The Cdc45 protein is ubiquitously distributed from yeast to human, supporting the notion that the formation of the CMG complex is universal in the eukaryotic DNA replication process. However, no archaeal homologue of Cdc45 has been identified until recently. A bioinformatic analysis showed that the primary structure of eukaryotic Cdc45 and prokaryotic RecJ share a common ancestry. Indeed, a homolog of the DNA binding domain of RecJ has been co-purified with GINS from S. solfataricus. Our experiment detected the stimulation of the 5’-3’ exonuclease activity of the RecJ homologs from P. furiosus and T. kodakarensis by the cognate GINS complexes. The RecJ homolog from T. kodakarensis forms a stable complex with the GINS, and the 5’-3’ exonuclease activity is enhanced in vitro; therefore, the RecJ homolog was designated as GAN, from GINS-associated nuclease in a recent paper. These current findings will promote further research on the structures and functions of the higher-order unwindosome in archaeal and eukaryotic cells.