The initiation of DNA replication requires dynamic biomolecular interactions, which are temporally and spatially regulated to allow genome duplication only once per cell cycle. During eukaryotic replication initiation, the MCM helicase is loaded as an inactive double hexamer encircling double-stranded DNA (dsDNA). It is activated by a set of proteins called firing factors in a kinase-dependent manner, thereby forming the CMG complex (Cdc45, MCM, GINS), which encircles single-stranded DNA (ssDNA) and thus can unwind dsDNA. Although the essential components for helicase activation are known, we do not understand the remarkable topological transition between the inactive helicase encircling dsDNA and the active helicase encircling ssDNA. For this to happen, the ring-shaped MCM helicase must open between two subunits in a regulated manner. Therefore, I aim to (1) uncover the trajectory of ssDNA ejection from the helicase central channel and (2) dissect the role of firing factors in helicase activation. The objectives of the proposal are to determine (i) which helicase subunit interface has to open to eject ssDNA, (ii) which region of helicase interacts with ssDNA during helicase activation, (iii) what is the topology of helicase activation intermediates and (iv) which firing factors interact with ssDNA during strand ejection. I will employ biochemistry with various crosslinking strategies combined with mass spectrometry to characterize the dynamics of protein-protein and protein-DNA interactions during helicase activation. Using cryogenic-Electron Microscopy (cryo-EM), I will investigate the structure of intermediates of helicase activation. MCM helicase subunits and firing factors are conserved from yeast to humans, and their increased expression is correlated with poor survival in cancer patients. Since flexible interfaces of protein-protein interactions are promising drug target, results obtained during this project will facilitate anticancer drug design.
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