At the intersection of DNA Replication and Genome integrity: Functional characterisation of the replicative helicase MCM2-7 during S-phase and replication stress.

DNA复制是所有生物体中的一个重要过程，当调控不当时，是基因组不稳定和癌症的主要原因。微染色体维持2-7（MCM 2 -7）复合物是一种环状异六聚体解旋酶，在细胞周期的G1期加载到DNA上，并保持无活性，直到细胞致力于DNA复制。然后，MCM 2 -7被激活，转化为CMG复合物（Cdc 45-MCM 2 -7-GINS），并从其核心挤出一条DNA链。因此，它在复制叉的尖端解开双链DNA（dsDNA）。在此过程中，MCM 2 -7提供单链DNA（ssDNA）作为DNA依赖性DNA聚合酶的模板。MCM 2 -7如何装载到dsDNA上并在复制起点被激活已被广泛研究，核心DNA复制过程已被纯化的蛋白质重建。然而，令人惊讶的是，很少有人知道的分子机制，使复制机制，以保持基因组的稳定性时，遇到的障碍，如自然路障，DNA损伤，或转录复制冲突。路障的例子是一般的转录因子和转录RNA聚合酶，它们是庞大的DNA结合蛋白质复合物。此外，拓扑应力，如R-环，可导致染色质不稳定。最后，DNA损伤对解旋酶提出了巨大的挑战，特别是单链断裂或链间交联。在所有这些情况下，CMG复合物必须克服DNA损伤，但旁路或暂停机制知之甚少。因此，这项研究的主要目的是揭示受调控的MCM 2 -7开环如何有助于细胞遗传互补的忠实复制的机制。使用最先进的技术，全基因组分析和化学生物学方法来控制细胞中的MCM 2 -7环开放，我将解决以下目标：1。在不存在和存在DNA损伤的情况下，在DNA合成期间对MCM 2 -7开环要求的阐明，以及2.）描述ssDNA和一种晚期复制激活蛋白Mcm 10在转录-复制冲突中的作用，将高分辨率测序技术与蛋白质组学相结合，将允许精确定义天然DNA结合的蛋白质复合物，并在DNA复制过程中MCM 2 -7环开放的背景下对其进行表征。本文提出的工作产生的结果将确定重塑解旋酶，改变染色质景观或去除RNA转录物以避免与癌症直接相关的复制应激的因素，因为DNA复制和复制应激中的缺陷会促进基因组不稳定性。此外，MCM 2 -7失调与许多恶性肿瘤有关。总之，该提案旨在系统地分析DNA复制和转录事件的交叉点，以MCM 2 -7环开放的新角度保护基因组完整性。

DNA Replication is an essential process in all living organisms, and when misregulated presents a major cause of genomic instability and cancer. The Mini Chromosome Maintenance 2-7 (MCM2-7) complex is a ring-shaped heterohexameric helicase, which is loaded onto DNA in G1-phase of the cell cycle and remains inactive until the cell commits to DNA replication. Then, MCM2-7 is activated, transformed into the CMG complex (Cdc45-MCM2-7-GINS) and extrudes a single DNA strand from its core. Consequently , it unwinds double-stranded DNA (dsDNA) at the tip of the replication fork. During this process, MCM2-7 provides single-stranded DNA (ssDNA) as a template for the DNA-dependent DNA polymerases. How MCM2-7 is loaded onto dsDNA and activated at replication origins has been widely studied, and the core DNA replication process has been reconstituted with purified proteins. However, surprisingly little is known about the molecular mechanisms that allow the replication machinery to preserve genomic stability when encountering obstacles such as natural roadblocks, DNA damage, or transcription-replication conflicts. Examples for roadblocks are general transcription factors and transcribing RNA polymerases, which are bulky, DNA-bound protein complexes. Furthermore, topological stresses, such as R-loops, can cause chromatin destabilisation. Finally, DNA damage presents a huge challenge for the unwinding helicase, particularly single strand breaks or interstrand crosslinks. In all these cases the CMG complex must overcome a DNA insult but bypassing or pausing mechanisms are only poorly understood. Therefore, the main objective of this research proposal is to uncover the mechanisms how regulated MCM2-7 ring opening contributes to faithful duplication of the genetic complement of the cell. Using state-of the-art techniques, genome-wide analyses, and a chemical-biology approach to control MCM2-7 ring opening in cells, I will tackle the following aims: 1.) Elucidation of MCM2-7 ring opening requirements during DNA synthesis in the absence and presence of DNA damage and 2.) Characterising the roles of ssDNA and Mcm10, a late replication activation protein, in transcription-replication conflicts.Combining high resolution sequencing techniques with proteomics will allow the precise definition of native DNA-bound protein complexes and their characterisation in context of MCM2-7 ring opening during DNA replication. The results generated by the work proposed here will identify factors that remodel the helicase, alter the chromatin landscape, or remove RNA transcripts to avoid replication stress with a direct relevance to cancer, as defects in DNA replication and replications stress promote genomic instability. Furthermore, MCM2-7 misregulation has been linked to many malignancies. In summary, this proposal aims to systematically analyse the intersection of DNA replication and transcription events that safeguard genome integrity with a new angle on MCM2-7 ring opening.