Project description
Imaging of chromatin duplication at high resolution
DNA replication is a tightly regulated process in order to prevent errors in the control mechanisms linked to the development of cellular abnormalities and genetic diseases, including the onset of cancer. Recent studies revealed distinct steps leading to the initiation of eukaryotic genome duplication. Structural investigations so far have involved the imaging of isolated replication steps using simplified templates of linear duplex DNA. However, the natural substrate of the eukaryotic replication machinery is a compact chromatin. The EU-funded CRYOREP project will apply newly developed protocols to perform biochemistry experiments in combination with cryo-electron microscopy with the aim of visualising chromatin duplication at high resolution.
Objective
In eukaryotic cells, DNA replication is tightly regulated to ensure that the genome is duplicated only once per cell cycle. Errors in the control mechanisms that regulate chromosome ploidy cause genomic instability, which is linked to the development of cellular abnormalities, genetic disease and the onset of cancer. Recent reconstitution experiments performed with purified proteins revealed that initiation of eukaryotic genome duplication requires three distinct steps. First, DNA replication start sites are identified and targeted for the loading of an inactive MCM helicase motor, which encircles the double helix. Second, MCM activators are recruited, causing duplex-DNA untwisting. Third, upon interaction with a firing factor, the MCM ring opens to eject one DNA strand, leading to unwinding of the replication fork and duplication by dedicated replicative polymerases. These three events are not understood at a molecular level. Structural investigations so far aimed at imaging artificially isolated replication steps and used simplified templates, such as linear duplex DNA to study helicase loading or pre-formed forks to understand unwinding. However, the natural substrate of the eukaryotic replication machinery is not DNA but rather chromatin, formed of nucleosome arrays that compact the genome. Chromatin plays important regulatory roles in all steps of DNA replication, by dictating origin start-site selection and stimulating replication fork progression. Only by studying chromatin replication, we argue, will we understand the molecular basis of genome propagation. To this end, we have developed new protocols to perform visual biochemistry experiments under the cryo-electron microscope, to image chromatin duplication at high resolution, frozen as it is being catalysed. Using these strategies we want to generate a molecular movie of the entire replication reaction. Our achievements will change the way we think about genome stability in eukaryotic cells.
Fields of science
Programme(s)
Funding Scheme
ERC-COG - Consolidator GrantHost institution
NW1 1AT London
United Kingdom