A novel human hexameric DNA helicase: expression, purification and characterization.
A novel human hexameric DNA helicase: expression, purification and characterization.
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DOI：
10.1093/nar/29.8.1733
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发表时间：
2001-04
影响因子：
14.9
通讯作者：
E. Biswas-Fiss;R. Nagele;S. Biswas
中科院分区：
文献类型：
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作者：
E. Biswas-Fiss;R. Nagele;S. Biswas
我们从 HeLa 细胞中克隆、表达并纯化了六聚体人类 DNA 解旋酶 (hHcsA)。序列分析表明，hHcsA 与来自酿酒酵母和秀丽隐杆线虫的 DNA 解旋酶基因具有很强的序列同源性，表明该基因似乎从酵母到人类都具有很好的保守性。 hHcsA 基因在大肠杆菌中克隆和表达，并纯化至均质。表达的蛋白质的亚基分子量为 116 kDa，通过尺寸排阻色谱分析其天然分子量表明 hHcsA 是六聚体蛋白质。 hHcsA 蛋白具有很强的 DNA 依赖性 ATP 酶活性，单链 DNA (ssDNA) 刺激该活性 >/=5 倍。人hHcsA解旋双链DNA并且易位极性分析表明DNA解旋的极性是在5'-->3'方向。解旋酶活性受到人和酵母复制蛋白 A 的刺激，但不受大肠杆菌 ssDNA 结合蛋白的显着刺激。我们使用原位杂交分析了 HeLa 细胞在细胞周期各个阶段的 hHcsA 基因的表达水平。我们的结果表明，从 mRNA 水平可以看出，hHcsA 基因的表达具有细胞周期依赖性。 hHcsA 表达的最高水平在 G(1) 晚期/S 早期观察到，表明该蛋白在 S 期和 DNA 合成中可能发挥作用。
We have cloned, expressed and purified a hexameric human DNA helicase (hHcsA) from HeLa cells. Sequence analysis demonstrated that the hHcsA has strong sequence homology with DNA helicase genes from Saccharomyces cerevisiae and Caenorhabditis elegans, indicating that this gene appears to be well conserved from yeast to human. The hHcsA gene was cloned and expressed in Escherichia coli and purified to homogeneity. The expressed protein had a subunit molecular mass of 116 kDa and analysis of its native molecular mass by size exclusion chromatography suggested that hHcsA is a hexameric protein. The hHcsA protein had a strong DNA-dependent ATPase activity that was stimulated >/=5-fold by single-stranded DNA (ssDNA). Human hHcsA unwinds duplex DNA and analysis of the polarity of translocation demonstrated that the polarity of DNA unwinding was in a 5'-->3' direction. The helicase activity was stimulated by human and yeast replication protein A, but not significantly by E.coli ssDNA-binding protein. We have analyzed expression levels of the hHcsA gene in HeLa cells during various phases of the cell cycle using in situ hybridization analysis. Our results indicated that the expression of the hHcsA gene, as evidenced from the mRNA levels, is cell cycle-dependent. The maximal level of hHcsA expression was observed in late G(1)/early S phase, suggesting a possible role for this protein during S phase and in DNA synthesis.