Komori K, Sakae S, Fujikane R, Morikawa K, Shinagawa H, Ishino Y
Department of Molecular Biology and Department of Structural Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
Nucleic Acids Res. 2000 Nov 15;28(22):4544-51. doi: 10.1093/nar/28.22.4544.
The Hjc protein of Pyrococcus furiosus is an endonuclease that resolves Holliday junctions, the intermediates in homologous recombination. The amino acid sequence of Hjc is conserved in Archaea, however, it is not similar to any of the well-characterized Holliday junction resolvases. In order to investigate the similarity and diversity of the enzymatic properties of Hjc as a Holliday junction resolvase, highly purified Hjc produced in recombinant Escherichia coli was used for detailed biochemical characterizations. Hjc has specific binding activity to the Holliday-structured DNA, with an apparent dissociation constant (K:(d)) of 60 nM. The dimeric form of Hjc binds to the substrate DNA. The optimal reaction conditions were determined using a synthetic Holliday junction as substrate. Hjc required a divalent cation for cleavage activity and Mg(2+) at 5-10 mM was optimal. Mn(2+) could substitute for Mg(2+), but it was much less efficient than Mg(2+) as the cofactor. The cleavage reaction was stimulated by alkaline pH and KCl at approximately 200 mM. In addition to the high specific activity, Hjc was found to be extremely heat stable. In contrast to the case of SULFOLOBUS:, the Holliday junction resolving activity detected in P. furiosus cell extract thus far is only derived from Hjc.
嗜热栖热菌的Hjc蛋白是一种核酸内切酶，可解析霍利迪连接体，即同源重组中的中间体。Hjc的氨基酸序列在古菌中保守，但与任何已充分表征的霍利迪连接体解离酶均不相似。为了研究Hjc作为霍利迪连接体解离酶的酶学性质的相似性和多样性，使用在重组大肠杆菌中产生的高度纯化的Hjc进行详细的生化表征。Hjc对霍利迪结构的DNA具有特异性结合活性，表观解离常数（Kd）为60 nM。Hjc的二聚体形式与底物DNA结合。以合成霍利迪连接体为底物确定了最佳反应条件。Hjc切割活性需要二价阳离子，5-10 mM的Mg2+最为适宜。Mn2+可替代Mg2+，但作为辅因子其效率远低于Mg2+。碱性pH和约200 mM的KCl可刺激切割反应。除了具有高比活性外，还发现Hjc具有极高的热稳定性。与硫磺菌的情况相反，迄今为止在嗜热栖热菌细胞提取物中检测到的霍利迪连接体解离活性仅源自Hjc。
