The Cdc6 nucleotide-binding site regulates its activity in DNA replication in human cells
The Cdc6 nucleotide-binding site regulates its activity in DNA replication in human cells
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DOI：
10.1091/mbc.10.8.2631
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发表时间：
1999-08-01
影响因子：
3.3
通讯作者：
Fanning, E
中科院分区：
文献类型：
--
作者：
Herbig, U;Marlar, CA;Fanning, E
芽殖酵母的Cdc6蛋白及其在其他物种中的同源物在DNA复制的起始中起着重要作用。已克隆编码Cdc6（HsCdc6）的人类同源物的cDNA，并以可溶性和功能活性形式表达为融合蛋白。纯化的蛋白质特异性结合ATP并缓慢水解，而HsCdc 6突变体在步行者A或B基序中含有氨基酸取代是有缺陷的。突变蛋白保留了结合HsOrc1和HsCdc6的能力，但在核苷酸存在下显示异常构象。在G1期将任一突变蛋白显微注射到人细胞中可抑制DNA复制，这表明ATP结合和HsCdc6的水解对DNA复制至关重要。
The Cdc6 protein of budding yeast and its homologues in other species play an essential role in the initiation of DNA replication. A cDNA encoding a human homologue of Cdc6 (HsCdc6) has been cloned and expressed as a fusion protein in a soluble and functionally active form. The purified protein bound specifically to ATP and slowly hydrolyzed it, whereas HsCdc6 mutants containing amino acid substitutions in the Walker A or B motifs were defective. The mutant proteins retained the ability to bind HsOrc1 and HsCdc6 but displayed aberrant conformations in the presence of nucleotides. Microinjection of either mutant protein into human cells in G1 inhibited DNA replication, suggesting that ATP binding and hydrolysis by HsCdc6 are essential for DNA replication.