Costa Alessandro, Pape Tillmann, van Heel Marin, Brick Peter, Patwardhan Ardan, Onesti Silvia
Division of Cell and Molecular Biology, Imperial College London SW7 2AZ, UK.
J Struct Biol. 2006 Oct;156(1):210-9. doi: 10.1016/j.jsb.2006.04.001. Epub 2006 May 2.
The primary candidate for the eukaryotic replicative helicase is the MCM2-7 complex, a hetero-oligomer formed by six AAA+ paralogous polypeptides. A simplified model for structure-function studies is the homo-oligomeric orthologue from the archaeon Methanothermobacter thermoautotrophicus. The crystal structure of the DNA-interacting N-terminal domain of this homo-oligomer revealed a double hexamer in a head-to-head configuration; single-particle electron microscopy studies have shown that the full-length protein complex can form both single and double rings, in which each ring can consist of a cyclical arrangement of six or seven subunits. Using single-particle techniques and especially multivariate statistical symmetry analysis, we have assessed the changes in stoichiometry that the complex undergoes when treated with various nucleotide analogues or when binding a double-stranded DNA fragment. We found that the binding of nucleotides or of double-stranded DNA leads to the preferred formation of double-ring structures. Specifically, the protein complex is present as a double heptamer when treated with a nucleotide analogue, but it is rather found as a double hexamer when complexed with double-stranded DNA. The possible physiological role of the various stoichiometries of the complex is discussed in the light of the proposed mechanisms of helicase activity.
真核生物复制解旋酶的主要候选者是MCM2-7复合物，它是由六个AAA+旁系同源多肽形成的异源寡聚体。用于结构功能研究的一个简化模型是嗜热自养甲烷杆菌的同源寡聚直系同源物。这种同源寡聚体与DNA相互作用的N端结构域的晶体结构揭示了一种头对头构型的双六聚体；单颗粒电子显微镜研究表明，全长蛋白质复合物可以形成单环和双环，其中每个环可以由六个或七个亚基的循环排列组成。使用单颗粒技术，特别是多变量统计对称分析，我们评估了该复合物在用各种核苷酸类似物处理或结合双链DNA片段时化学计量的变化。我们发现，核苷酸或双链DNA的结合导致双环结构的优先形成。具体而言，当用核苷酸类似物处理时，蛋白质复合物以双七聚体形式存在，但当与双链DNA复合时，它更常以双六聚体形式存在。根据所提出的解旋酶活性机制，讨论了该复合物各种化学计量可能的生理作用。
