Escherichia coli DNA helicase II is active as a monomer
Escherichia coli DNA helicase II is active as a monomer
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DOI：
10.1074/jbc.274.18.12488
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发表时间：
1999-04-30
影响因子：
4.8
通讯作者：
Matson, SW
中科院分区：
文献类型：
--
作者：
Mechanic, LE;Hall, MC;Matson, SW
解旋酶被认为以寡聚体（通常是二聚体或六聚体）的形式发挥作用，在这里我证明，虽然大肠杆菌 DNA 解旋酶 II (UvrD) 能够二聚化，如酵母双杂交系统、凝胶过滤色谱和平衡沉降超速离心 (K-d = 3.4 mu M) 中的正相互作用所证明的那样，但该蛋白质在体内具有活性 在体外，作为单体，缺乏 C 端 40 个氨基酸的突变体 (UvrD Delta 40C) 未能二聚化，但在 ATP 水解和解旋酶测定中与野生型蛋白一样活跃。此外，uvrD Delta 40C等位基因完全补充了嘧啶二聚体甲基定向错配修复和切除修复中解旋酶II的缺失，使用野生型UvrD和失活UvrD点突变体的生化抑制实验为功能性单体提供了进一步的证据。这项研究首次直接证明了活性单体解旋酶，并提出了单体 DNA 解旋的模型。
Helicases are thought to function as oligomers (generally dimers or hexamers), Here me demonstrate that although Escherichia coli DNA helicase II (UvrD) is capable of dimerization as evidenced by a positive interaction in the yeast two-hybrid system, gel filtration chromatography, and equilibrium sedimentation ultracentrifugation (K-d = 3.4 mu M), the protein is active in vivo and in vitro as a monomer, A mutant lacking the C-terminal 40 amino acids (UvrD Delta 40C) failed to dimerize and yet was as active as the wild-type protein in ATP hydrolysis and helicase assays. In addition, the uvrD Delta 40C allele fully complemented the loss of helicase II in both methyl-directed mismatch repair and excision repair of pyrimidine dimers, Biochemical inhibition experiments using wild-type UvrD and inactive UvrD point mutants provided further evidence for a functional monomer. This investigation provides the first direct demonstration of an active monomeric helicase, and a model for DNA unwinding by a monomer is presented.