酿酒酵母DNA复制起始过程中解旋酶Mcm2-7的变构激活机理

Saccharomyces cerevisiae is one of the best model organisms for studying DNA replication, a fundamental and essential process in all living things. In eukaryotes, DNA helicase Mcm2-7, the core replication component, is assembled as an inactive double hexameric form in G1 phase. It remains to be elucidated how it is activated when cells enter S phase to initiate bidirectional replication. In our recently published work, we developed a novel strategy to purify the native endogenous MCM supercomplexes instead of the conventional in vitro reconstitution. We were thus able to identify the new components and dynamics of MCM supercomplexes during cell cycle progression, which leads to a Mcm10-interaction dependent “MCM double hexamer split model”. Based on these findings, particularly the well-established approaches for studying native replication machines, we commitment to investigate the detailed mechanisms of MCM remodeling and activation prior to replication initiation. Moreover, we will explore the possible crosstalk or cooperation between Mcm10 and other initiation factors. This study will help to resolve the long-lasting puzzles of replicative helicase MCM and replication initiation in eukaryotes, which will inevitably contribute to our knowledge of genome stability and cellular growth.

酿酒酵母是研究DNA复制这一生命科学前沿课题的主要模式。真核复制机器的核心组分解旋酶Mcm2-7在G1期以无活性的双六聚体构象组装，但在S期如何被激活起始复制的机制不明。本课题组2015年发表的工作中，采用不同于前人体外重建的新策略，建立了一整套直接纯化酵母内源Mcm2-7及其超级蛋白复合体的新方法，揭示了MCM复合体的新组分及其内部构象随细胞周期行进的动态时空变化，首次提出了依赖Mcm10动态互作的“MCM双六聚体分离变构模型”。基于这一新的关键步骤的发现，尤其是已成熟的天然复制机器研究体系，我们急需获得重点资助，得以更深入地探究MCM变构激活及复制起始的完整工作机制，并综合分析各激活通路之间可能存在的关联合作机制。本研究将有助于解决困扰真核DNA复制领域数十年之久的MCM活性及起始控制这一重点难题，使经典的DNA双向复制模型更加完整清晰，加深对遗传信息稳定传递及生物生长增殖过程的认识。

真核复制机器的核心组分解旋酶Mcm2-7在G1期以无活性的双六聚体构象组装，但在S期如何被激活起始复制的机制不明。本项目以模式生物酿酒酵母为材料,首次在S期鉴定到同时包含两个MCM、两个Cdc45和两个GINS的 “double CMG (d-CMG)”复合物。d-CMG在S期开始形成积累，在S期后期逐渐减少直至消失；d-CMG没有解旋酶活性和DNA复制能力。通过电镜观察到CMG相关复合体中有多种不同构象的d-CMG存在，为无活性的MCM双六聚体向有活性的单CMG/replisome转换过程中的一系列中间态复合体。更进一步，Mcm10-Mcm2-7具有相互作用，且受细胞周期及S-CDK磷酸化调控；Mcm10与DDK协同作用促进Mcm2-7的激活；Mcm10促进MCM双六聚体分离变构。这些进展为20多年前Stillman等人提出的起始前复合物 (pre-IC) 概念提供了生化内涵，使经典的DNA双向复制模型更加完整清晰，加深了对遗传信息稳定传递及生物生长增殖过程的认识。