A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase
A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase
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DOI：
10.1073/pnas.030539597
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发表时间：
2000-02-15
影响因子：
11.1
通讯作者：
Stillman, B
中科院分区：
文献类型：
--
作者：
Chong, JPJ;Hayashi, MK;Stillman, B
小染色体维持蛋白（MCM）是真核生物DNA复制所必需的。到目前为止，所有真核生物都含有六种高度相关的mcm，它们显然在DNA复制中共同起作用。对嗜热古细菌热自养甲烷杆菌的全基因组测序使我们仅鉴定出一个mcm样基因（ORF Mt1770）。该基因与真核细胞中的MCM4最为相似。在这里，我们表达并纯化了嗜热养结核分枝杆菌MCM蛋白。纯化后的蛋白形成一个复合体，其分子质量约为850 kDa，与双六聚体的形成一致。该蛋白具有atp独立的DNA结合活性，dma刺激的atp酶活性，可区分单链和双链DNA，以及链位移（解旋酶）活性，可解开多达500个碱基对。3‘到5’解旋酶的活性需要ATP水解和一个功能性的核苷酸结合位点。此外，双六聚体形式是活性解旋酶，因此MCM复合体很可能在真核生物和古细菌中充当复制DNA解旋酶，古细菌中简化的复制机制可能为真核生物DNA复制起始所需机制的组装提供了简化的模型。
The minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. Thus far, all eukaryotes have been shown to contain six highly related MCMs that apparently function together in DNA replication. Sequencing of the entire genome of the thermophilic archaeon Methanobacterium thermo-autotrophicum has allowed us to identify only a single MCM-like gene (ORF Mt1770). This gene is most similar to MCM4 in eukaryotic cells. Here we have expressed and purified the M. thermoaototrophicum MCM protein. The purified protein forms a complex that has a molecular mass of approximate to 850 kDa, consistent with formation of a double hexamer, The protein has an ATP-independent DNA-binding activity, a DMA-stimulated ATPase activity that discriminates between single- and double-stranded DNA, and a strand-displacement (helicase) activity that can unwind up to 500 base pairs. The 3' to 5' helicase activity requires both ATP hydrolysis and a functional nucleotide-binding site. Moreover, the double hexamer form is the active helicase, It is therefore likely that an MCM complex acts as the replicative DNA helicase in eukaryotes and archaea, The simplified replication machinery in archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication.