Postdoctoral Training Fellow
This is a full time, fixed term (4 years) position, with possibility of extension, on Crick terms and conditions of employment.
The research group
The laboratory focuses on the mechanism of chromosome replication using biochemical reconstitution, light and cryo-electron microscopy (cryo-EM).
Using yeast and human proteins, we study the initiation step of DNA replication, which is highly regulated to ensure that every chromosome is copied only once per cell cycle (1). It involves a cascade of molecular events reflected in discrete structural intermediates, such as helicase loading (2), phosphorylation (3), DNA melting (4) and replication fork establishment (5).
We currently focus on how DNA unwinding and synthesis are coordinated, and how the replication machinery efficiently replicates DNA molecules that are packaged in nucleosome arrays (6).
To perform our research, we develop new tools in three areas.
1. Kinetics. We pioneered time resolved electron microscopy approaches, enabling us to describe the sequential, concerted mechanism for loading pairs of helicase molecules onto origin DNA (2).
2. Image analysis. We develop RECONSIL, a pipeline to extract information on physiological context from cryo-electron micrographs, which is otherwise lost in single-particle image processing (7).
3. Correlative microscopy. We develop methods to follow DNA replication with single-molecule fluorescence and electron microscopy using one cryo-EM grid support.
More information can be found on the Francis Crick Institute web page:
or our lab website
Please direct any questions to Alessandro Costa (firstname.lastname@example.org).
1 Costa, A. & Diffley, J. F. X. The Initiation of Eukaryotic DNA Replication. Annual Review of Biochemistry, (2022).
2 Miller, T. C. R., Locke, J., Greiwe, J. F., Diffley, J. F. X. & Costa, A. Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM. Nature 575, 704-710, (2019).
3 Greiwe, J. F., … Diffley JF.X. & Costa, A. Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase. Nature Structural & Molecular Biology 29, 10-20, (2022).
4 Lewis, J. S., … Diffley JF.X. & Costa, A. Mechanism of replication origin melting nucleated by CMG helicase assembly. Nature 606, 1007-1014, (2022).
5 Douglas, M. E., Ali, F. A., Costa, A. & Diffley, J. F. X. The mechanism of eukaryotic CMG helicase activation. Nature 555, 265-268, (2018).
6 Willhoft, O. & Costa, A. A structural framework for DNA replication and transcription through chromatin. Current Opinion in Structural Biology 71, 51-58, (2021).
7 Puhringer, T., Greiwe, J. F., Miller, T. C. R. & Costa, A. ReconSil: An electron microscopy toolbox to study helicase function at an origin of replication. Methods in Enzymology 672, 203-231, (2022).
The replication machinery is powered by a helicase motor that unwinds the double helix and replicative polymerases that duplicate DNA. How are DNA unwinding and synthesis coordinated? How is continuous DNA synthesis on the leading strand coupled with discontinuous Okazaki fragment synthesis on the lagging strand? How are nucleosomes that protect parental DNA uncoiled and how are they re-assembled on duplicated DNA?
To address these questions the postdoctoral training fellow will perform in vitro reconstitution of chromatin replication reactions using established protocols. Reactions will be imaged by cryo-EM for single particle analysis using state of the art instruments available at the Crick Institute. These include a Thermofisher Talos Arctica and a Glacios microscopes equipped with Falcon 3 detectors and two Titan Krios equipped with Falcon 4 detectors and Selectris Imaging filter. Computing facilities include the Crick CPU and GPU clusters, as well as stand alone workstations available in the lab. To monitor the kinetics of chromatin replication, single-molecule fluorescence imaging will be employed using correlative microscopy approaches recently established in our group. Three-dimensional structures will be solved and atomic models built to understand replication mechanisms at the molecular level. Structural mechanisms will be validated using site-directed mutagenesis and biochemical assays.
Postdoctoral Training Fellows are expected to lead their own projects and contribute to other studies by working together with other members of the lab or external collaborators.
Key experience and competencies
This position is ideal for a biochemist or single-molecule fluorescence microscopist who wants to learn cryo-EM. Alternatively, this position would suit cryo-EM scientists interested in developing their biochemistry and working on correlative light and electron microscopy, at the single molecule level.
- PhD in Biochemistry (and related fields) or in the final stages of PhD submission
- Expertise at least one of three areas: protein biochemistry, structural biology or single-molecule fluorescence microscopy.
- Research articles published or submitted for publication in peer-reviewed journals.
- Experience of experimental design.
- Ability to work independently and as part of a team.
- Experience in the biochemistry of nucleic acid interacting proteins.
- Experience in single-particle cryo-EM and atomic-model building.
- Expertise in single-molecule fluorescence microscopy.