Skip to main content
European Commission logo
English English
CORDIS - EU research results
CORDIS
CORDIS Web 30th anniversary CORDIS Web 30th anniversary

Unravelling the mechanism of eukaryotic helicase activation

Project description

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.

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.

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
Total cost
€ 212 933,76