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Visualising chromatin replication with cryo-electron microscopy

  • Full or part time
    Dr A Costa
  • Application Deadline
    Tuesday, November 12, 2019
  • Funded PhD Project (Students Worldwide)
    Funded PhD Project (Students Worldwide)

Project Description

This 4-year PhD studentship is offered in Dr Alessandro Costa’s Group based at the Francis Crick Institute (the Crick).

Eukaryotic chromosomes are packaged in nucleosome arrays that protect and regulate our genome. In each nucleosome, DNA wraps around a proteinaceous core formed of eight histones, which contain covalent modifications, called “epigenetic marks”. These marks modulate gene expression, which is essential for the cellular response to environmental cues and organismal development.

Our group studies chromosome replication, which is catalysed by a molecular machine named the replisome. Within the replisome, an ATP-fuelled molecular motor unwinds the double helix, providing the template three replicative DNA polymerases. These polymerases specifically copy the leading or the lagging strand at the replication fork, resulting in accurate DNA duplication, and the maintenance of genome stability that is key for healthy cell proliferation [1]. As a result of chromosome replication, not only genetic but also epigenetic information is inherited. Therefore, while copying DNA, the replisome must be able to disassemble nucleosomes found ahead of the replication fork, and recycle parental histones onto newly duplicated DNA. Specific pathways exist for the selective deposition of histones onto leading or lagging strand DNA, however the molecular mechanism is unknown [2].

We employ a combination of biochemistry and single-particle cryo-electron microscopy (EM) to study chromosome replication. Using near-atomic resolution imaging and single-particle reconstruction, we reconstitute entire chromatin duplication reactions in silico, as they occur in a test tube. Using these tools, we have recently understood how the helicase, which opens the DNA at the replication fork, is initially loaded onto its substrate, and how it harnesses the energy derived from ATP binding and hydrolysis to unwind the double helix [3]. We have obtained the first high-resolution view of the leading-strand polymerase bound to the helicase [4], and were the first lab to use cryo-EM to capture an enzyme as it changes the structure of a nucleosome, to facilitate genome propagation [5]. Our new PhD student will build on our progress to study the mechanism of parental histone re-deposition onto duplicated DNA. The successful candidate will learn to assemble the replisome on nucleosome-decorated DNA, and run replication reactions to image histone-recycling events. To do this, the student will leverage the lab’s expertise in the biochemistry of chromatin and molecular machines, and as well as in cryo-EM. The end result of this study will be the generation of a molecular movie of chromatin replication, where individual frames correspond to reaction intermediates described at high-resolution. These studies will significantly advance our understanding of epigenetic inheritance, genome stability maintenance and the strategies developed in eukaryotes to avoid malignant cellular transformations and cancer.

This project would suit candidates with a background in biochemistry and a passion for molecular mechanisms. Although experience in Structural Biology and computing would be an advantage, it is not a requirement. Most importantly, we are looking for an inventive and self-motivated individual who enjoys interacting with colleagues around the lab and in our institute. By joining our lab, the student will become part of a friendly group of 10 researchers from 8 different countries.

Applicants should hold or expect to gain a first/upper second-class honours degree or equivalent in a relevant subject and have appropriate research experience as part of, or outside of, a university degree course and/or a Masters degree in a relevant subject.

APPLICATIONS MUST BE MADE ONLINE VIA OUR WEBSITE (ACCESSIBLE VIA THE ‘APPLY NOW’ LINK ABOVE) BY 12:00 (NOON) 13 NOVEMBER 2019. APPLICATIONS WILL NOT BE ACCEPTED IN ANY OTHER FORMAT.

Funding Notes

Successful applicants will be awarded a non-taxable annual stipend of £22,000 plus payment of university tuition fees. Students of all nationalities are eligible to apply.

References

1. Pellegrini, L. and Costa, A. (2016)
New insights into the mechanism of DNA duplication by the eukaryotic replisome.
Trends in Biochemical Sciences 41: 859-871. PubMed abstract
2. Miller, T. C. and Costa, A. (2017)
The architecture and function of the chromatin replication machinery.
Current Opinion in Structural Biology 47: 9-16. PubMed abstract
3. Douglas, M. E., Abid Ali, F., Costa, A. and Diffley, J. F. X. (2018)
The mechanism of eukaryotic CMG helicase activation.
Nature 555: 265-268. PubMed abstract
4. Goswami, P., Abid Ali, F., Douglas, M. E., Locke, J., Purkiss, A., Janska, A., . . . Costa, A. (2018)
Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome.
Nature Communications 9: 5061. PubMed abstract
5. Maskell, D. P., Renault, L., Serrao, E., Lesbats, P., Matadeen, R., Hare, S., . . . Cherepanov, P. (2015)
Structural basis for retroviral integration into nucleosomes.
Nature 523: 366-369. PubMed abstract



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