Identification of a novel helicase activity unwinding branched DNAs from the hyperthermophilic archaeon, Pyrococcus furiosus
Identification of a novel helicase activity unwinding branched DNAs from the hyperthermophilic archaeon, Pyrococcus furiosus
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DOI：
10.1074/jbc.m413417200
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发表时间：
2005-04-01
影响因子：
4.8
通讯作者：
Ishino, Y
中科院分区：
文献类型：
--
作者：
Fujikane, R;Komori, K;Ishino, Y
为了确定古细菌的分支迁移活性，我们用几种色谱法分离了火球菌细胞提取物，并测定了合成Holliday连接的atp依赖性分辨率。在柱馏分中确定了目标活性，并利用部分纯化馏分确定了分支迁移活性的最佳反应条件。通过筛选富氏疟原虫基因组DNA构建的热稳定蛋白文库，成功克隆出相应的基因。这个名为hjm （Holliday junction migration）的基因编码一种由720个氨基酸组成的蛋白质。Hjm蛋白在古细菌中是保守的，属于解旋酶超家族2。同源性研究表明，Hjm蛋白与人类Pol Theta、HEL308和果蝇Mus308蛋白序列相似，参与DNA修复，而在细菌和酵母中未发现相似的序列。hhm解旋酶可能在极端环境下生物体的修复系统中起着核心作用。
To identify the branch migration activity in archaea, we fractionated Pyrococcus furiosus cell extracts by several chromatography and assayed for ATP-dependent resolution of synthetic Holliday junctions. The target activity was identified in the column fractions, and the optimal reaction conditions for the branch migration activity were determined using the partially purified fraction. We successfully cloned the corresponding gene by screening a heat-stable protein library made by P. furiosus genomic DNA. The gene, hjm (Holliday junction migration), encodes a protein composed of 720 amino acids. The Hjm protein is conserved in Archaea and belongs to the helicase superfamily 2. A homology search revealed that Hjm shares sequence similarity with the human Pol Theta, HEL308, and Drosophila Mus308 proteins, which are involved in a DNA repair, whereas no similar sequences were found in bacteria and yeast. The Hjm helicase may play a central role in the repair systems of organisms living in extreme environments.