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

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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.