Job Description

Salary for this Role:

From \xc2\xa341,935, subject to skills and experience

Job Title:

Postdoctoral Fellow

Reports to: John Diffley

Closing Date: 30/Sept/2023 23.59 GMT

:

Postdoctoral Fellow \xe2\x80\x93 Diffley lab

Reporting to: John Diffley, Associate Research Director

Contact term: This is a full-time, fixed term 4-year position on Crick terms and conditions of employment.

Please note, there is another Postdoctoral Fellow position being advertised in Diffley Lab in parallel to this advert if this is of interest as well.

The Research Group

The Chromosome Replication Laboratory at the Francis Crick Institute aims to understand the mechanism and regulation of eukaryotic DNA replication and its misregulation in human cancer [1]. We study this process using biochemistry, genetics and cell biology in both budding yeast and humans. We focus on several different, but related topics.

The Mechanism of Initiation

The initiation of DNA replication occurs in two steps in eukaryotes. The MCM helicase is loaded at replication origins during G1 phase of the cell cycle in an inactive form as a head to head double hexamer and then converted converted to the active CMG helicase during S phase. We have developed a variety of approaches to study this process based on the full reconstitution of DNA replication with purified proteins [2-4]. We also have a long-standing collaboration with Alessandro Costa\xe2\x80\x99s Macromolecular Machines Laboratory at the Crick [5-8] on the analysis of DNA replication using cryo electron microscopy.

The Regulation of Initiation and its Misregulation in Cancer

We have contributed over the years to understanding of how replication initiation is regulated by protein kinases including cyclin dependent kinase (CDK), Dbf4 dependent kinase (DDK) and the DNA damage checkpoint kinase Rad53. CDK plays two opposing roles in initiation: It inhibits MCM loading and is essential for MCM activation. Consequently, MCM loading is limited to G1 phase when CDK activity is low. We are especially interested in understanding how compromising this low CDK period by deregulating CDK activity leads to replicative stress and genome doubling [9].

Mechanisms of Chromatin Replication

We have reconstituted complete chromatin replication [2] and we are using this together with newly developed nanopore sequencing approaches to understand how parental nucleosomes are specifically inherited to the leading and lagging strands during replication and how this is coordinated with deposition of newly synthesised histones.

Role of the DNA Damage Checkpoint in Regulating DNA Replication Forks

We have previously shown that the Rad53 protein kinase plays a crucial role in somehow stabilising stalled DNA replication fork [10,11]. We have recently found checkpoint dependent inhibition of replication origin firing [12, 13] together with checkpoint dependent reduction of replication fork speed via Mrc1 [14] act to prevent excess Okazaki fragment synthesis. These excess Okazaki fragments act to sequester the processive DNA synthesis machinery, and prevent restart of leading strand replication.

Further details about the Chromosome Replication Laboratory and links to other Crick-related information can be found at: https://www.crick.ac.uk/research/labs/john-diffley

Publications

1. Costa, A. and J.F.X. Diffley, The Initiation of Eukaryotic DNA Replication. Annu Rev Biochem, 2022. 91: p. 107-131.

2. Kurat, C.F., et al., Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates. Mol Cell, 2017. 65(1): p. 117-130.

3. Yeeles, J.T., et al., Regulated eukaryotic DNA replication origin firing with purified proteins. Nature, 2015. 519(7544): p. 431-5.

4. Yeeles, J.T., et al., How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication. Mol Cell, 2017. 65(1): p. 105-116.

5. Douglas, M.E., et al., The mechanism of eukaryotic CMG helicase activation. Nature, 2018. 555(7695): p. 265-268.

6. Greiwe, J.F., et al., Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase. Nat Struct Mol Biol, 2022. 29(1): p. 10-20.

7. Lewis, J.S., et al., Mechanism of replication origin melting nucleated by CMG helicase assembly. Nature, 2022. 606(7916): p. 1007-1014.

8. Miller, T.C.R., et al., Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM. Nature, 2019. 575(7784): p. 704-710.

9. Zeng, J., et al., Cyclin E-induced replicative stress drives p53-dependent whole-genome duplication. Cell, 2023. 186(3): p. 528-542 e14.

10. Tercero, J.A. and J.F.X. Diffley, Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature, 2001. 412(6846): p. 553-7.

11. Tercero, J.A., M.P. Longhese, and J.F.X. Diffley, A central role for DNA replication forks in checkpoint activation and response. Mol Cell, 2003. 11(5): p. 1323-36.

12. Santocanale, C. and J.F.X. Diffley, A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature, 1998. 395(6702): p. 615-8.

13. Zegerman, P. and J.F.X. Diffley, Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation. Nature, 2010. 467(7314): p. 474-8.

14. McClure, A.W. and J.F. Diffley, Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation. Elife, 2021. 10.

The Project

Much of our past work has used purified proteins from budding yeast. We have recently reconstituted the loading of the MCM double hexamer onto DNA using purified human proteins. This opens the door to explore a new system, highly relevant for human health. The purpose of this role will be to use these loaded MCM double hexamers as a starting point to reconstitute the initiation of replication with purified human proteins.

In this project, some of the specific aims include but are not limited to:

Establishment of the MCM loading reaction

Purification of the firing factors involved in initiating replication

Use of these factors to establish replication initiation, using a deliberate, stepwise approach

Collaborate to identify and characterise intermediates in this reaction using cryo-EM

Characterise the essential CDK and DDK phosphorylation events in this process

The Diffley lab strives to foster an environment that welcomes, includes, and values people with diverse backgrounds and experiences. We provide all Postdoctoral Fellows with the support, space, and resources they need to pursue their goals and place and emphasis on furthering their careers. They will lead their own projects, contribute to other projects on a collaborative basis (both in the lab and with external collaborators). The ability to work in a team is essential.

Responsibilities of the Postdoctoral Fellow include the following:

Undertake academic research and develop projects in a timely manner

Maintain clear records of all experiments in electronic format

Contribute ideas to the research programme

Adapt existing and develop new scientific techniques and experimental protocols

Use specialist scientific equipment in a laboratory environment

Acquire, analyse, and review scientific data to test and refine working hypotheses

Provide guidance and training to less experienced members of the research group

Contribute to the preparation of scientific reports and journal articles

Collaborate with colleagues in Crick research groups and Science Technology Platforms

Attend and participate in academic activities such as lab meetings, journal clubs, wider network meetings, and retreats

These duties are a guide to the work that the post holder will be required to undertake and may change with scientific developments.

Key experience and competencies

The post holder should embody and demonstrate our core Crick values:

Bold; Open; Collegial

Essential:

PhD (awarded or expected) in some field related to Biomedicine or Biology including but not limited to biochemistry, genetics, cell biology, or chemistry.

A demonstrated ability to generate and pursue independent research ideas

A demonstrated ability to work as part of an interdisciplinary team

Willingness to mentor junior members of the laboratory

Strong communication and presentation skills

Strong organisational and record-keeping skills

Track record of scientific scholarship, evidenced by preprints, publications or submitted manuscripts in refereed journals

Desired:

Experience in protein purification, especially from yeast or baculovirus expression

Experience with some complex biochemical assays.

Experience with close collaborations.

About Us

At the Crick, we conduct research at the forefront of biomedical research. We combine rigour with an open and collaborative culture, and are outward-looking, reflecting our status as a partnership of six organisations aiming to pool knowledge, ideas and resources.

We have a wide research portfolio with no divisions or departments, bringing biomedical researchers together with clinicians, physical scientists and applied scientists from our pharmaceutical partners.

We aim to attract the most talented researchers and support them to tackle innovative research questions. Our science technology platforms provide our researchers with access to state-of-the-art technology and expertise.

We provide an excellent learning environment with dedicated education programmes in public engagement with science, education and personal development, and a postdoc training programme that prepares scientists for leadership roles in science.

If you are interested in applying for this role, please apply via our .

The closing date for applications is 15 September 2023 at 23:59

All offers of employment are subject to successful security screening and continuous eligibility to work in the United Kingdom.

If you require a visa to work in the UK we will help support your application should you be successful

Find out what benefits the Crick has to offer:

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Equality, Diversity & Inclusion:

We welcome applications from all backgrounds. We are committed to providing equal employment opportunities, regardless of ethnicity, nationality, gender, sexual orientation, gender identity, religion, pregnancy, age, disability, or civil partnership, marital or family status. We particularly welcome applications from people who are Minority Ethnic as they are currently underrepresented in the Crick at this level.

Diversity is essential to excellence in scientific endeavour. It increases breadth and perspective, leading to more innovation and creativity. We want the Crick to be a place where everyone feels valued and where diversity is celebrated and seen as part of the foundation for our Institute\xe2\x80\x99s success.

The Crick is committed to creating equality of opportunity and promoting diversity and inclusivity. We all share in the responsibility to actively promote dignity, respect, inclusivity and equal treatment and it is our aim to ensure that these principles are reflected and implemented in all strategies, policies and practices.

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