Project description DEENESFRITPL Mechanism of helicase activation in the initiation of eukaryotic DNA replication The eukaryotic DNA replication starts with minichromosome maintenance (MCM) helicase encircling double-stranded DNA (dsDNA) as an inactive double hexamer. The activation is driven by the kinase dependent formation of the Cdc45 MCM-GINS protein complex, which encircles single stranded DNA (ssDNA) and unwinds dsDNA. Funded by the Marie Skłodowska-Curie Actions programme, the MechHelicaseActiv8on project will investigate the topological transition between the inactive and active helicase states, when the ring-shaped MCM helicase opens between two subunits in a regulated manner. Using various crosslinking strategies combined with mass spectrometry and cryogenic electron microscopy, the project aims to study the trajectory of ssDNA ejection from the helicase central channel and the role of the protein complex in helicase activation. Show the project objective Hide the project objective Objective 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. Fields of science natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsproteomicsmedical and health sciencesbasic medicinemedicinal chemistrynatural sciencesbiological sciencesgeneticsDNAmedical and health sciencesclinical medicineoncologynatural scienceschemical sciencesanalytical chemistrymass spectrometry Keywords DNA replication protein-protein interactions nucleoprotein complexes helicases Cryo-EM Crosslinking Mass Spectrometry replication initiation Programme(s) H2020-EU.1.3. - EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions Main Programme H2020-EU.1.3.2. - Nurturing excellence by means of cross-border and cross-sector mobility Topic(s) MSCA-IF-2020 - Individual Fellowships Call for proposal H2020-MSCA-IF-2020 See other projects for this call Funding Scheme MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF) Coordinator THE FRANCIS CRICK INSTITUTE LIMITED Net EU contribution € 212 933,76 Address 1 MIDLAND ROAD NW1 1AT London United Kingdom See on map Region London Inner London — West Camden and City of London Activity type Research Organisations Links Contact the organisation Opens in new window Website Opens in new window Participation in EU R&I programmes Opens in new window HORIZON collaboration network Opens in new window Total cost € 212 933,76
