Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus

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 ∼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.

激烈热球菌（Pyrococcus furiosus）的Hjc蛋白是一种可解离霍利迪联结体（Holliday junction）的内切核酸酶，而霍利迪联结体是同源重组过程中的关键中间产物。古菌（Archaea）中Hjc的氨基酸序列具有保守性，但其与已充分表征的各类霍利迪联结体解离酶均无同源性。为探究作为霍利迪联结体解离酶的Hjc其酶学性质的相似性与多样性，研究人员采用重组大肠杆菌（Escherichia coli）表达纯化得到的高纯度Hjc蛋白，开展了详尽的生化特性表征。Hjc对霍利迪结构DNA具有特异性结合活性，其表观解离常数（Kd）为60 nM。Hjc的二聚体形式可与底物DNA结合。研究以合成型霍利迪联结体为底物，确定了该酶的最优反应条件：Hjc的切割活性依赖二价阳离子，其中5~10 mM的Mg²+为最优辅因子浓度；Mn²+可替代Mg²+作为辅因子，但催化效率远低于Mg²+。碱性pH环境与约200 mM的KCl可促进该切割反应。除具备较高的比活性外，Hjc还具有极强的热稳定性。与硫化叶菌（Sulfolobus）的情况不同，目前在激烈热球菌细胞提取物中检测到的霍利迪联结体解离活性仅来源于Hjc。