Project description
Visualisation of origin-dependent DNA replication on native substrates
Errors in the mechanisms that control DNA replication can cause genomic instability and lead to the development of genetic diseases and cancer. Structural studies have focused on isolated replication complexes using simplified DNA substrates for imaging DNA unwinding and replisome architecture. Funded by the Marie Skłodowska-Curie Actions programme, the DNA-Rep-EM project will integrate single-particle cryo-electron microscopy with biochemical approaches to obtain images of origin-dependent DNA replication reactions in vitro on native DNA substrates. The researchers will establish short origin-containing DNA substrates for the loading of a single bidirectional replication fork and the visualisation of large numbers of protein-bound origins within a single field of view.
Objective
Genome duplication is essential for cell proliferation. Errors in the mechanisms that control DNA replication can cause genomic instability and lead to the development of genetic diseases and cancer. In vitro reconstitution of DNA replication with purified yeast proteins has helped uncover important mechanisms of DNA replication. How changes in protein structure regulates function during key events in origin activation and replisome progression, such as melting of the DNA duplex upon CMG helicase assembly, or the mode of binding of Pol alpha during primer synthesis remain unknown. To date, structural studies have focused on imaging artificially isolated replication complexes using simplified DNA substrates to understand DNA unwinding and replisome architecture. To truly understand the mechanisms that control DNA replication, future structural studies must not only visualise isolated complexes, but also reconstituted reactions. To address this issue, I will integrate single-particle cryo electron-microscopy (cryo-EM) with sophisticated biochemical approaches to image origin-dependent DNA replication reactions in vitro on native DNA substrates. To do so, I will establish short origin-containing DNA substrates that permit loading of a single bidirectional replication fork, allowing large numbers of protein bound origins to be visualised within a single field of view. Initially, I will investigate how the structure of duplex DNA changes upon CMG formation during origin activation. Next, I will examine the molecular mechanisms of primer synthesis after origin activation using both cryo-EM and in vitro DNA replication reactions. Finally, I will capture and image synthesising intact replisomes at near atomic resolution. By visualising entire DNA replication reactions instead of isolated replication complexes at high resolution, we will gain a deeper understanding of the molecular mechanisms that permit the eukaryotic replisome to function during genome duplication.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences genetics DNA
- natural sciences biological sciences biochemistry biomolecules proteins
- medical and health sciences clinical medicine oncology
- natural sciences biological sciences genetics genomes
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Keywords
Project’s keywords as indicated by the project coordinator. Not to be confused with the EuroSciVoc taxonomy (Fields of science)
Project’s keywords as indicated by the project coordinator. Not to be confused with the EuroSciVoc taxonomy (Fields of science)
Programme(s)
Multi-annual funding programmes that define the EU’s priorities for research and innovation.
Multi-annual funding programmes that define the EU’s priorities for research and innovation.
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H2020-EU.1.3. - EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions
MAIN PROGRAMME
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H2020-EU.1.3.2. - Nurturing excellence by means of cross-border and cross-sector mobility
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Topic(s)
Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.
Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.
Funding Scheme
Funding scheme (or “Type of Action”) inside a programme with common features. It specifies: the scope of what is funded; the reimbursement rate; specific evaluation criteria to qualify for funding; and the use of simplified forms of costs like lump sums.
Funding scheme (or “Type of Action”) inside a programme with common features. It specifies: the scope of what is funded; the reimbursement rate; specific evaluation criteria to qualify for funding; and the use of simplified forms of costs like lump sums.
MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)
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Call for proposal
Procedure for inviting applicants to submit project proposals, with the aim of receiving EU funding.
Procedure for inviting applicants to submit project proposals, with the aim of receiving EU funding.
(opens in new window) H2020-MSCA-IF-2020
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Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.
NW1 1AT London
United Kingdom
The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.