Descripción del proyecto
El mecanismo de activación de la helicasa durante el inicio de la replicación del ADN eucariota
La replicación del ADN eucariota comienza con la helicasa de mantenimiento de microcromosomas (MCM), que envuelve el ADN bicatenario (ADNbc) como un doble hexámero inactivo. La activación está impulsada por la formación dependiente de cinasa del complejo proteico Cdc45 MCM-GINS, que rodea el ADN monocatenario (ADNmc) y desenrolla el ADNbc. En el proyecto MechHelicaseActiv8on, financiado por las Acciones Marie Skłodowska-Curie, se investigará la transición topológica entre los estados inactivos y activos de la helicasa, cuando la helicasa de MCM anular se abre entre dos subunidades de manera regulada. El objetivo del equipo del proyecto es estudiar la trayectoria de eyección del ADNmc desde el canal central de la helicasa, así como el papel del complejo proteico en la activación de la helicasa. Para ello, se emplearán varias estrategias de entrecruzamiento combinadas con espectrometría de masas y microscopía electrónica criogénica.
Objetivo
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.
Ámbito científico
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsproteomics
- medical and health sciencesbasic medicinemedicinal chemistry
- natural sciencesbiological sciencesgeneticsDNA
- medical and health sciencesclinical medicineoncology
- natural scienceschemical sciencesanalytical chemistrymass spectrometry
Palabras clave
Programa(s)
Régimen de financiación
MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Coordinador
NW1 1AT London
Reino Unido