| Summary: | This thesis provides new insights into the recruitment mechanisms of factors to replicative origins that are important for helicase activation during initiation of DNA replication in budding yeast. DNA replication origins are recognised by the Origin Recognition Complex (ORC), which recruits, during late M-phase, Cdc6, Cdt1 and MCM2-7 to form a pre-replication complex (pre-RC). MCM2-7 represents the core of the eukaryotic replicative helicase, but it is inactive within the pre-RC. In early S-phase, the pre-RC is converted into the pre-initiation complex (pre-IC), leading to stable Cdc45/GINS/MCM2-7 (CMG) complex formation and helicase activation. This process depends on the recruitment of a series of factors (Sld3, Cdc45, Sld2, Sld7, Dpb11 ...) and is regulated by kinases (DDK and S-CDK) but their specific function and mechanism of action is largely unknown. A systematic pairwise interaction analysis was performed using purified and in vitro translated proteins from Saccharomyces cerevisiae in order to understand how these factors interact with each other and MCM2-7, allowing helicase loading and activation. During this study a new network of interactions was identified, which is required for the stable recruitment of Cdc45 to MCM2-7. This was concomitant with the in vitro reconstitution of Cdc45 loading with purified proteins, which described Sld2, in addition to Sld3, as Cdc45 loading factor. Moreover, the Mcm2 subunit of the MCM2-7 hexamer was identified as a central player in Cdc45 recruitment, representing a binding surface for Sld2, Sld3 and Cdc45. The interaction sites in these proteins were mapped and mutants were generated to disrupt the formation of the complex containing Cdc45. The results obtained in this work constitute a comprehensive overview on the interactions occurring during pre-RC, and CMG formation. Additionally, they provide important details on the mechanisms leading to helicase activation and contribute to the understanding of this still poorly defined step of DNA replication.
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