Projektbeschreibung
Mechanismus der Helikaseaktivierung bei Einleitung der eukaryotischen DNA-Replikation
Die eukaryotische DNA-Replikation beginnt mit der MCM-Helikase (minichromosome maintenance), welche die doppelsträngige DNA (dsDNA) als inaktives Doppelhexamer umschließt. Die Aktivierung wird durch die kinaseabhängige Bildung des Cdc45 MCM-GINS-Proteinkomplexes ausgelöst, der DNA-Einzelstränge (ssDNA) umschließt und dsDNA abwickelt. Das im Rahmen der Marie-Skłodowska-Curie-Maßnahmen finanzierte Projekt MechHelicaseActiv8on wird den topologischen Übergang zwischen dem inaktiven und dem aktiven Helikasezustand untersuchen, bei dem sich die ringförmige MCM-Helikase zwischen zwei Untereinheiten auf regulierte Weise öffnet. Unter Einsatz verschiedener Vernetzungsstrategien in Kombination mit Massenspektrometrie und Kryoelektronenmikroskopie wird das Projekt die Bahn des ssDNA-Ausstoßes aus dem zentralen Helikasekanal und die Funktion des Proteinkomplexes bei der Helikaseaktivierung erforschen.
Ziel
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.
Wissenschaftliches Gebiet
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsproteomics
- medical and health sciencesbasic medicinemedicinal chemistry
- natural sciencesbiological sciencesgeneticsDNA
- medical and health sciencesclinical medicineoncology
- natural scienceschemical sciencesanalytical chemistrymass spectrometry
Schlüsselbegriffe
Programm/Programme
Thema/Themen
Aufforderung zur Vorschlagseinreichung
Andere Projekte für diesen Aufruf anzeigenFinanzierungsplan
MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Koordinator
NW1 1AT London
Vereinigtes Königreich