MechHelicaseActiv8on · Unravelling the mechanism of eukaryotic helicase activation
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2022-09-01 → 2024-08-31
- EU contribution
- €212,934
- Participants
- 1
- Scheme
- MSCA-IF
Lines connect the coordinator with its partners.
Results in brief
Unravelling the mechanism of eukaryotic helicase activation
The project aimed to unravel the mechanism of eukaryotic helicase activation in DNA replication initiation. During DNA replication, helicase is essential for unwinding the DNA double helix, a step critical for cellular division. However, the exact mechanisms by which the helicase is activated to start DNA unwinding, including the transition from double-stranded to single-stranded DNA, remain unclear. Understanding DNA replication mechanisms is fundamental to advancing knowledge in genome maintenance. Errors in DNA replication can lead to genetic mutations, contributing to cancer and other genetic disorders. By shedding light on helicase activation, this research has the potential to aid in the development of targeted therapies for diseases that result from DNA replication errors. The main objectives of the project were: (1) to uncover the fundamental mechanisms by which helicase is activated to initiate DNA unwinding, (2) to investigate the specific roles of various protein components within the helicase complex.
Data: CORDIS, © European Union
Project 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.
Original text from CORDIS.
Participants
- THE FRANCIS CRICK INSTITUTE LIMITED · LondonCoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union