Biochemistry & Proteomics Cell Cycle & Chromosomes Imaging Model Organisms Structural Biology & Biophysics
A 2023 Crick PhD project with Alessandro Costa
Project background and description
DNA replication is essential for the propagation of life. Errors in this process lead to genomic instability, which is a hallmark of cancer cells. DNA replication is performed by the replisome, a large molecular assembly containing a ring-shaped helicase that unwinds the double helix, and a set of replicative polymerases that copy DNA. Thanks to biochemical reconstitution efforts, we now know that a set of two replicative helicases are loaded onto DNA in an inactive form and, upon recruitment of a number of ‘firing factors’, the helicase unwinds the double helix, providing the single-stranded template for the replicative polymerases . The Molecular Machines laboratory has pioneered time-resolved cryo-EM approaches to visualise stages of the DNA replication reaction as they occur in a test tube, in three dimension and at near atomic resolution. Using this strategy, we explained how two helicases can be loaded sequentially onto origin DNA, forming a dimer that encircles the double helix . Our results explain how the symmetry of bidirectional replication is established. We then started to characterise the molecular mechanism of origin activation. This is a multistep process involving the selective phosphorylation of the helicase dimer , and the ATP-controlled nucleation of origin DNA melting .
The PhD student will build on our achievements to reconstitute the DNA replication process in a test tube, adapting existing biochemical protocols to cryo-EM imaging. Reconstituted reactions will be frozen on a cryo-EM grid at various points in time, to characterise the molecular events leading to replication fork establishment. Cryo-EM structures of key intermediates on the path to origin activation will be determined and the derived molecular mechanisms will be tested functionally. These efforts will allow generating a high-resolution molecular movie of DNA replication initiation.
This project will suit candidates with a background in Biochemistry and Structural Biology and a passion for molecular mechanism. While performing this work, the student will acquire expertise in molecular biology and biochemistry. The student will also development of time-resolved cryo-EM assays, and receive training in cryo-EM imaging and three-dimensional structure determination.
1. Costa, A. and Diffley, J.F.X. (2022)
The initiation of eukaryotic DNA replication.
Annual Review of Biochemistry 91: 107-131. PubMed abstract
2. Miller, T.C.R., Locke, J., Greiwe, J.F., Diffley, J.F.X. and Costa, A. (2019)
Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM.
Nature 575: 704-710. PubMed abstract
3. Greiwe, J.F., Miller, T.C.R., Locke, J., Martino, F., Howell, S., Schreiber, A., . . . Costa, A. (2022)
Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase.
Nature Structural & Molecular Biology 29: 10-20. PubMed abstract
4. Lewis, J.S., Gross, M.H., Sousa, J., Henrikus, S.S., Greiwe, J.F., Nans, A., . . . Costa, A. (2022)
Mechanism of replication origin melting nucleated by CMG helicase assembly.
Nature 606: 1007-1014. PubMed abstract