Graduate Program in Genetics, Molecular and Cell Biology, University of Southern California, Los Angeles, CA 90089, USA.
BMC Biochem. 2013 Feb 27;14:5. doi: 10.1186/1471-2091-14-5.
The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA. The ring-shaped Mcm2-7 hexamers comprising one of each subunit show helicase activity in vitro, and form double-hexamers on DNA. The Mcm4/6/7 also forms a hexameric complex with helicase activity in vitro.
We used an Escherichiai coli expression system to express various domains of Schizosaccharomyces pombe Mcm4, 6 and 7 in order to characterize their domain structure, oligomeric states, and possible inter-/intra-subunit interactions. We also successfully employed a co-expression system to express Mcm4/6/7 at the same time in Escherichiai coli, and have purified functional Mcm4/6/7 complex in a hexameric state in high yield and purity, providing a means for generating large quantity of proteins for future structural and biochemical studies.
Based on our results and those of others, models were proposed for the subunit arrangement and architecture of both the Mcm4/6/7 hexamer and the Mcm2-7 double-hexamer.
真核生物微小染色体维持（MCM）蛋白的异六聚体在基因组 DNA 的复制中起着至关重要的作用。由每个亚基组成的环形 Mcm2-7 六聚体在体外具有解旋酶活性，并在 DNA 上形成双六聚体。Mcm4/6/7 也在体外与具有解旋酶活性的六聚体复合物形成。
我们使用大肠杆菌表达系统来表达酿酒酵母 Mcm4、6 和 7 的各种结构域，以表征它们的结构域结构、寡聚状态以及可能的亚基间/内相互作用。我们还成功地在大肠杆菌中同时表达 Mcm4/6/7 的共表达系统，并以高产量和纯度纯化出具有功能的 Mcm4/6/7 六聚体复合物，为未来的结构和生化研究提供了大量蛋白质的生成方法。
基于我们的结果和其他人的结果，提出了 Mcm4/6/7 六聚体和 Mcm2-7 双六聚体的亚基排列和结构模型。
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