Characterization of the enzymatic activity of hChIR1, a novel human DNA helicase
Characterization of the enzymatic activity of hChIR1, a novel human DNA helicase
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DOI：
10.1093/nar/28.4.917
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发表时间：
2000-02-15
影响因子：
14.9
通讯作者：
Lahti, JM
中科院分区：
文献类型：
--
作者：
Hirota, Y;Lahti, JM
最近，我们克隆了两个高度相关的人类基因HCHLR1（DDX11）和HCHLR2（DDX12），它们似乎是酿酒酵母CHL1基因的同源物。核苷酸序列分析表明，这些基因编码DEAH家族的新成员：DNA解放酶，而CHL1的酶促活性尚未表征，但维持高富度富裕性染色体分离酵母中的蛋白质是必需的。在这里，我们报告说，HCHLR1蛋白是一种新型的人DNA解旋酶，我们使用杆菌病毒系统表达并纯化了HCHLR1，并分析了其酶促活性。重组HCHLR1蛋白具有严格依赖DNA，二价阳离子和ATP的ATPase和DNA解旋酶活性，这些活性被ATP结合域中的单个氨基酸取代所取消。 HCHLR1蛋白可以放弃DNA/DNA和RNA/DNA底物。它偏爱在短的单链DNA模板上在5' - > 3'方向上运动，但是，与其他DNA解旋酶不同，HCHLR1 DNA旋转酶可以在两个方向上沿单个方向的单链DNA转运，而当底物的底物非常非常长单链DNA区域。 HCHLR1的酶促活性表明，DNA解旋酶维持染色体分离的保真度是必需的。
Recently, we cloned two highly related human genes, hChlR1 (DDX11) and hChlR2 (DDX12), which appear to be homologs of the Saccharomyces cerevisiae CHL1 gene. Nucleotide sequence analysis suggests that these genes encode new members of the DEAH family of:DNA helicases, While the enzymatic activity of CHL1 has not been characterized, the protein is required for the maintenance of high fidelity chromosome segregation in yeast. Here we report that the hChlR1 protein is a novel human DNA helicase, We have expressed and purified hChlR1 using a baculo-virus system and analyzed its enzymatic activity. The recombinant hChlR1 protein possesses both ATPase and DNA helicase activities that are strictly dependent on DNA, divalent cations and ATP, These activities are abolished by a single amino acid substitution in the ATP-binding domain. The hChlR1 protein can unwind both DNA/DNA and RNA/DNA substrates. It has a preference for movement in the 5' --> 3' direction on short single-stranded DNA templates, However, unlike other DNA helicases, the hChlR1 DNA helicase can translocate along single-stranded DNA in both directions when substrates have a very long single-stranded DNA region. The enzymatic activities of hChlR1 suggest that DNA helicases are required for maintaining the fidelity of chromosome segregation.