Probing the function of individual Cyclin/Cdk complexes during the cell cycle

Background: Cyclin subunits, in combination with their Cyclin-dependent kinase (Cdk) domains, regulate many aspects of cell cycle progression, including DNA replication (Cyclin A2) and cell division (Cyclin B1). Cyclin A2 and Cyclin B1 share the same catalytic subunits, Cdk1 and Cdk2, therefore it is difficult to separate their functions because Cdk1 or Cdk2 inhibitors affect multiple Cyclin/Cdk complexes at once. For example, Cyclin B/Cdk1 is known to stimulate mitotic entry but Cyclin A2/Cdk1 could well play an important undiscovered role. The aim of this PhD is to use cutting-edge techniques in genome engineering to develop new ways to modify the endogenous localisation and activity of specific Cyclin/Cdk complexes. These tools will then be used to ask novel questions about the role of individual Cyclin/Cdk complexes during the cell cycle.

Methods: We have recently established genome-engineering methods to tag endogenous proteins in mammalian cells (using TALENs, see review1) and genetic mouse models to study Cdk’s2. We have successfully tagged endogenous Cyclin B1 in multiple different mammalian cell types and we have also recently established a method to relocalise Cyclin B1 using small molecule drugs – by forcing the dimerization of FRB and FKBP domains using rapamycin (fig.1). The PhD student will combine these methods to tag endogenous Cyclin A2 or Cyclin B1 complexes with an EYFP-FKBP domain (termed Fluorescent REcruitment DOMain or FREDOM). This will then be used to relocalise endogenous Cyclin/Cdk complexes or to develop ways to specifically inhibit Cyclin A2/Cdk1 or Cyclin B1/Cdk1 complexes (by using the tagged Cyclin subunit to recruit inhibitory Cdk1 peptides).

Aims:

1) To tag endogenous Cyclin A2 and Cyclin B1 with a FREDOM tag in different mammalian cell types

2) To develop the FREDOM system to inhibit specific Cyclin/Cdk complexes

3) To screen for specific Cyclin A2/Cdk1 and Cyclin B/Cdk1 substrates

4) To investigate the role of Cyclin A2/Cdk1 during mitotic entry

5) To investigate the role of protein localisation on Cyclin A2/Cdk and Cyclin B1/Cdk signalling

References

1. Joung, J.K. & Sander, J.D. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14, 49-55 (2013).

2. Lim, S. & Kaldis, P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development 140, 3079-93 (2013).

Supervisor: 
Dr Adrian Saurin
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A* Applicants only

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Eligible PhD students from the University of Dundee will spend 1 to 2 years of their PhD at an A*STAR research institute under joint supervision of staff at the University of Dundee and an A*STAR research institute