Chong J P, Hayashi M K, Simon M N, Xu R M, Stillman B
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1530-5. doi: 10.1073/pnas.030539597.
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 thermoautotrophicum 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. thermoautotrophicum MCM protein. The purified protein forms a complex that has a molecular mass of approximately 850 kDa, consistent with formation of a double hexamer. The protein has an ATP-independent DNA-binding activity, a DNA-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.
微小染色体维持（MCM）蛋白对于真核生物中的DNA复制至关重要。到目前为止，所有真核生物都已被证明含有六个高度相关的MCM蛋白，它们显然在DNA复制中共同发挥作用。嗜热古菌嗜热自养甲烷杆菌全基因组的测序使我们仅鉴定出一个类似MCM的基因（开放阅读框Mt1770）。该基因与真核细胞中的MCM4最为相似。在此，我们表达并纯化了嗜热自养甲烷杆菌的MCM蛋白。纯化后的蛋白形成了一个分子量约为850 kDa的复合物，这与形成双六聚体一致。该蛋白具有不依赖ATP的DNA结合活性、一种能区分单链和双链DNA的DNA刺激的ATP酶活性，以及一种能解开多达500个碱基对的链置换（解旋酶）活性。3'到5'解旋酶活性既需要ATP水解，也需要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复合物很可能在真核生物和古菌中充当复制性DNA解旋酶。古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一个简化模型。
