Description du projet
Mécanisme d’activation de l’hélicase dans l’initiation de la réplication de l’ADN eucaryote
La réplication de l’ADN eucaryote commence avec l’hélicase MCM (Minichromosome Maintenance) qui encercle l’ADN double brin (ADNdb) sous la forme d’un double hexamère inactif. L’activation est pilotée par la formation, sous la dépendance d’une kinase, du complexe protéique Cdc45 MCM-GINS, qui encercle l’ADN simple brin (ADNsb) et déroule l’ADNdb. Financé par le programme Actions Marie Skłodowska-Curie, le projet MechHelicaseActiv8on étudiera la transition topologique entre les états inactif et actif de l’hélicase lorsque l’hélicase MCM en forme d’anneau s’ouvre entre deux sous-unités de manière régulée. En utilisant diverses stratégies de réticulation combinées à la spectrométrie de masse et à la cryo-microscopie électronique, le projet entend étudier la trajectoire de l’éjection de l’ADNsb à partir du canal central de l’hélicase et le rôle du complexe protéique dans l’activation de l’hélicase.
Objectif
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.
Champ scientifique
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsproteomics
- medical and health sciencesbasic medicinemedicinal chemistry
- natural sciencesbiological sciencesgeneticsDNA
- medical and health sciencesclinical medicineoncology
- natural scienceschemical sciencesanalytical chemistrymass spectrometry
Mots‑clés
Programme(s)
Régime de financement
MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Coordinateur
NW1 1AT London
Royaume-Uni