Functional analysis of the MCM2-7 DNA entry/exit gate during S-phase

这项研究计划的主要目标是揭示受调控的MCM 2 -7开环如何影响复制解旋酶激活、DNA合成和DNA修复，这些将共同揭示一个对基因组稳定性至关重要的全新途径。精确的DNA复制是所有活细胞的基本标志。因此，真核细胞已经进化出一个复杂的系统，以确保准确的染色体复制。通过将DNA复制分为两个步骤，细胞确保其遗传物质在每个细胞周期仅复制一次。在第一步中，MCM 2 -7（微型染色体维持）六聚体组装在双链起始DNA周围的大分子环结构中，促进解旋酶加载到DNA复制起点上。该复合物在早期S期的第二步中通过磷酸化和与许多复制因子的相互作用被激活，导致一条DNA链从其环中挤出，但这一步骤的分子细节仍然难以捉摸。在这个项目中，我将使用一种独家可用的化学生物学方法，允许在MCM 2 -7复合物内控制解旋酶界面的闭合，本论文的主要目的是：1）确定MCM 2 - 7的DNA出口门; 2）确定激活复制解旋酶的蛋白质复合物;和3）理解在不存在和存在DNA损伤的情况下在DNA合成期间潜在的MCM 2 -7开环要求。DFG对这项开创性的跨学科研究提案的资助将使我能够剖析DNA复制中一种全新的机制，即受调控的MCM 2 -7开环。这项工作将对DNA复制和DNA修复的多个方面具有重要意义。此外，该项目还可能成为未来开发具有抗癌活性的化疗药物的基础，因为抑制MCM 2 -7开环提供了一种有效的途径来阻断癌细胞的增殖。

The main objective of this research proposal is to uncover how regulated MCM2-7 ring opening affects replicative helicase activation, DNA synthesis and DNA repair, which together will unravel a completely new pathway that is essential for genome stability.Precise DNA replication is an essential hallmark of all living cells. Thus, eukaryotic cells have evolved a sophisticated system to ensure accurate chromosome duplication. By dividing DNA replication into two steps, cells ensure that their genetic material is replicated only once per cell cycle. In the first step, the MCM2-7 (mini chromosome maintenance) hexamer is assembled in a macromolecular ring structure around double-stranded origin DNA, promoting helicase loading onto origins of DNA replication. This complex gets activated during the second step in early S-phase by phosphorylation and the interaction with a number of replication factors, resulting in extrusion of one DNA-strand from its ring, but the molecular details of this step remained elusive.In this project I will use an exclusively available, chemical biology approach allowing the controlled closure of a helicase interface within the MCM2-7 complex, which was previously used to identify the DNA entry gate of MCM2-7.Using state-of-the-art techniques, I want to tackle the following three key aims: 1) Identification of the DNA exit gate of MCM2-7; 2) Characterisation of the protein complexes that activate the replicative helicase; and 3) Comprehension of potential MCM2-7 ring opening requirements during DNA synthesis in the absence and presence of DNA damage. Funding of this pioneering and interdisciplinary research proposal by the DFG will allow me to dissect a completely novel mechanism in DNA replication, the regulated MCM2-7 ring opening. This work will have major relevance for multiple aspects of DNA replication and DNA repair. In addition, the project may also form the basis for future chemotherapeutics development with anti-cancer activity, as the inhibition of MCM2-7 ring opening offers an efficient route to block proliferation of cancer cells.