Biochemical characterization of the Hjc Holliday junction resolvase of <i>Pyrococcus furiosus</i>

作     者：Komori, K Sakae, S Fujikane, R Morikawa, K Shinagawa, H Ishino, Y 

作者机构：BERI Dept Mol Biol Suita Osaka 5650874 Japan BERI Dept Biol Struct Suita Osaka 5650874 Japan Osaka Univ Microbial Dis Res Inst Dept Mol Microbiol Suita Osaka 5650871 Japan 

出 版 物：《NUCLEIC ACIDS RESEARCH》 (Nucleic Acids Res.)

年 卷 期：2000年第28卷第22期

页      面：4544-4551页

核心收录：

学科分类：0710[理学-生物学] 08[工学] 09[农学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

主　　题：古细菌蛋白质类/遗传学 古细菌蛋白质类/分离和提纯 古细菌蛋白质类/代谢 阳离子 二价/药理学 DNA/化学 DNA/遗传学 DNA/代谢 脱氧核糖核酸酶类 位点特异性Ⅱ型/遗传学 脱氧核糖核酸酶类 位点特异性Ⅱ型/分离和提纯 脱氧核糖核酸酶类 位点特异性Ⅱ型/代谢 内切脱氧核糖核酸酶类/遗传学 内切脱氧核糖核酸酶类/分离和提纯 内切脱氧核糖核酸酶类/代谢 酶稳定性 霍利迪连结体解离酶类 氢离子浓度 动力学 镁/药理学 锰/药理学 核酸构象 氯化钾/药理学 蛋白质结合/药物作用 火球菌属/酶学 底物特异性 

摘      要：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 nan, The dimeric form of Hjc binds to the substrate DMA. The optimal reaction conditions were determined using a synthetic Holliday junction as substrate, Hjc required a divalent cation for cleavage activity and Mg2+ at 5-10 mM was optimal, Mn2+ could substitute for Mg2+, but it was much less efficient than Mg2+ as the cofactor. The cleavage reaction was stimulated by alkaline pH and KCI at similar to 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 *** cell extract thus far is only derived from Hjc.