DNA解旋酶MCM双六聚体构象的形成机制与生理功能

MCM（Mini-chromosome maintenance）protein is essential for genome replication in all eukaryotes, which plays important roles both as the licensing factor during initiation and as the coordinator between replication fork movement and the cell cycle progression. Recent studies showed that MCM might form double hexamers (DH) in vivo and in vitro besides its traditional single hexamer ring shape. What's the physiological function of such particular conformation of MCM remains elusive. The mechanism to form doule-hexamer structure may be conserved among archaeal and eukaryotic MCM proteins through our previous phylogenetic tree analysis. Hyperthermophilic Sulfolobus islandicus becomes an ideal model organism because this genetically tractable species contains only one MCM protein, which makes it possible to carry out the structural and functional analysis for homo-oligomer of MCM. We'll investigate the ability and mechanism of SisMCM to form double hexamers through combination of approaches as Electro-microscopy and mutation assays. The effects of the DH formation on its unwinding activity will be measured. The results from SisMCM will be further applied to MCM2-7 of Saccharomyces cerevisiae, in which multiple mutations can be easily achieved. By taking advantage of both Sulfolobus and budding yeast models，it might be possible to answer how and why MCM complex should be assembled as the DH form. Our studies may contribute to dissolve the enigmas as "MCM paradox".

MCM（Mini-chromosome maintenance）蛋白被普遍认为是所有真核生物DNA复制所必需的DNA解旋酶，不仅仅在DNA复制起始的控制、复制叉的组装及其行进过程起着重要作用，还参与基因组复制与细胞周期之间的偶联调控。最近一些研究发现MCM蛋白除了经典的六聚体环状结构外，还可能形成双六聚体等构象。MCM为什么形成双六聚体构象？这种构象与MCM活性有什么关系？前期已经获得的MCM分子进化树等结果已暗示极端嗜热冰岛硫化叶菌与酵母中存在保守的双六聚体形成机制。本项目将同时采用这两种各具优势的模式材料，首先对由单亚基组成的冰岛硫化叶菌SisMCM同聚体运用遗传、生化与电镜等手段解析双六聚体构象形成机制、动态变化及其生理功能；然后在酵母MCM2-7六个亚基中相应的保守功能域进行突变或互补分析，探讨MCM蛋白构象与其解旋酶活性之间的关系，将有助于解决"MCM悖论"等悬而未决的问题。

MCM（Mini-chromosome maintenance）蛋白被普遍认为是所有真核生物DNA 复制所必需的DNA 解旋酶，不仅仅在DNA 复制起始的控制、复制叉的组装及其行进过程起着重要作用，还参与基因组复制与细胞周期之间的偶联调控。最近一些研究发现MCM 蛋白除了经典的六聚体环状结构外，还可能形成双六聚体等构象。本项目针对真核基因组复制领域中的前沿难题——MCM 解旋酶的结构与功能的动态调控，主要研究了MCM蛋白形成双六聚体（Double Hexamers）与其解旋功能之间的关系。我们报道古菌MCM同源蛋白存在甲基化修饰，证明甲基化修饰增强了高温环境下MCM解旋酶的活性。报道了Sld3与Mcm2-7复合体中的Mcm2/6两个亚基之间的直接相互作用，鉴定了互作关键氨基酸残基，突变分析证明Sld3-MCM互作是MCM激活过程中必需的一个中间环节。更重要的是，本项目研究中发现了一个与MCM双六聚体特异相互作用的蛋白，Mcm10。建立了在酵母细胞内分析MCM双六聚体形成-分离的新方法，证明Mcm10是首个参与MCM双六聚体分离的必需因子。这些结果深化了我们对真核生物DNA复制起始过程中MCM双六聚体形成-分离-激活过程的认识。本项目相关成果以通讯或共通讯作者在Cell Reports, PLoS Genetics, EMBO Reports, Frontiers in Microbiology等生物学专业期刊发表论文9篇。