PRIMASE_NMR · Visualizing Primase Initiating DNA Replication using NMR Spectroscopy
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2016-03-01 → 2018-02-28
- EU contribution
- €175,420
- Participants
- 1
- Scheme
- MSCA-IF
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Results in brief
Visualizing Primase Initiating DNA Replication using NMR Spectroscopy
The genetic information encoded in DNA in a fundamentally important element of any living organism. A key aspect of DNA is its capability to be replicated, allowing for the transfer of the genetic information upon generations of cells or organisms. It is therefore of uttermost importance to understand the detailed mechanism of DNA replication. DNA replication happens in three steps. First, the helicases unwind the DNA to make it accessible to other biological actors. Then, primases interact with the DNA strands to synthesize short primers that will finally be extended by polymerases to copy the full genetic information. DNA is made of building blocks, the nucleotides, and DNA transcription corresponds to assemble nucleotides in the correct order to ensure the exact duplication of the initial DNA strand. Polymerases are able to add an extra nucleotide to an existing DNA strand but primases have the unique ability to assemble nucleotides from scratch and in particular to assemble together the two first nucleotides. This step of initiation is absolutely key in the DNA replication process as any further duplication procedures rely on this initial step. However, up to now, the detailed mechanism by which primases act remains quite elusive. To understand how the initiation of replication appends, it is necessary to understand how those nucleotides interact with the primase at the atomic level. To do so, we used Nuclear Magnetic Resonance (NMR) spectroscopy, the technique of choice to study biomolecular transient interactions at the atomic resolution. Using NMR spectroscopy our objectives are to understand: (i) how the primase and the DNA interact with nucleotides, (ii) how this interactions influence the properties of the primase, the DNA and the nucleotides, (iii) how it is possible to create the first bond between two nucleotides and (iv) how this dinucleotides can be extended into a short primer of defined length. Answering those questions will have a strong impact on our fundamental understanding of DNA replication. Due to the essential role of DNA replication, the better understanding of this process will help in the development of novel biological or biomedical techniques related to this mechanism.
Data: CORDIS, © European Union
Project objective
The replication of DNA and therefore of the genetic information is realized at the molecular level in three steps: helicases open the DNA, primases initiate the replication and polymerases use this primer to duplicate the DNA strand. The precise mechanism of action of primases has up to now remain quite elusive and thus a better understanding of how primase works would be of great interest to fully understand the process of DNA replication. Primases are classed into two groups, bacterial and archeal/eukaryal, and some archeal primase have the particularity to carry their biological function without requiring the association in a larger molecular complex, making them an accessible target to Nuclear Magnetic Resonance (NMR) spectroscopy.In this project we propose to exploit the unique capacity of NMR spectroscopy for determining structure and dynamics of biomolecules in solution to investigate the ORF904 primase, free and in complex with its DNA template and the cofactors necessary for primer synthesis. We aim to characterize how this system assembles and to provide an atomic resolution picture of its mechanism of action, just before the creation of the first phosphodiester bound that initiate primer formation. We will also investigate the conformational changes occurring during the primer synthesis using Electron Paramagnetic Resonance (EPR) spectroscopy. Spectroscopic and computational innovative approaches will be developed to describe this complex dynamic system and complementary integrative structural biology will be used to support our findings.By this study we aim to provide an accurate description of a primase accomplishing its biological function and therefore significantly deepen our knowledge of DNA replication. This in turn could be used in cancer biology to develop new therapeutic approaches.
Original text from CORDIS.
Participants
- EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH · ZuerichCoordinatorSwitzerland
Links
Data: CORDIS, © European Union