Primary Supervisor: Dr Aga Gambus, Institute of Cancer and Genomic Sciences
Secondary supervisor: Dr Marco Saponaro
PhD project title: Regulation of replication machinery disassembly
University of Registration: University of Birmingham
Cell division requires accurate duplication of all genetic information. DNA replication is precisely regulated as unrepaired errors can lead to severe consequences, such as genetic disease, cancer and premature ageing. Surprisingly, despite six decades of research little was known about the termination stage of eukaryotic replication. The Gambus lab recently provided a much-needed breakthrough in this field and since then has characterised the first elements of the replisome disassembly mechanism. Moreover, preliminary data from the Gambus lab indicate that perturbations of replisome disassembly can lead to DNA damage. The perfect execution of the termination stage of DNA replication is therefore equally essential as initiation and elongation for maintenance of the stability of our genomes. However, our knowledge of this process is very limited.
Using Xenopus laevis egg extract model system, we discovered that one subunit of the replicative helicase, Mcm7, is polyubiquitylated at termination by the Cul2Lrr1 ubiquitin ligase (Lrr1 is a substrate specific receptor for the ligase), promoting removal of the helicase from chromatin in a p97/VCP segregase-dependent manner. As the helicase forms the organising centre of the replisome, helicase removal leads to its disassembly. Importantly, this process is evolutionarily conserved in C.elegans and S.cerevisiae. Moreover, our work has demonstrated the existence of a back-up pathway of replisome disassembly in mitosis driven by a different ubiquitin ligase TRAIP. Together with data from two other labs, these findings led to the first ever model of eukaryotic replication termination, describing an elegant and highly regulated process. However, much is still to be learned about its execution and coordination.
Our extensive mass spectrometry analyses of the post-termination replisome and whole chromatin (CHROMASS) upon inhibition of replisome dissolution provides us with potential candidates for other modifying enzymes or factors regulating replisome disassembly or fork termination. To investigate the role of these candidates, we will follow our tested path of delivery. We will prepare bespoke antibodies against all four proteins (Xenopus studies depend on custom-made antibodies, for which we have over 13 years’ experience and an excellent track record in raising), study their association with chromatin and terminated replisomes, and immunodeplete from egg extract to examine their role in termination. We will express corresponding proteins (in E.coli) and purify catalytically-dead mutants, and study their effect on replisome disassembly. Finally, we will take advantage of existing small molecule inhibitors against some of the candidates and study the effects of inhibition of these proteins on replisome disassembly.
As replisome disassembly is the last step of the termination process, its inhibition is a good readout for screening of factors involved in termination of replication forks, but it does not specify which step of the process the factor is involved in. To distinguish whether the role is in convergence of replication forks or replisome disassembly, we will visualise DNA structures and terminating replication forks upon inhibition or immunodepletion of chosen factors using electron microscopy and single molecule visualisation of replisation forks terminating.
As soon as we identify a factor that does affect replisome disassembly and/or fork convergence, the student will follow up this factor to understand in depth the mechanism of its action. The studies will be performed to start with in Xenopus laevis egg extract, but the findings obtained will be then translated to human immortalised cell lines.
- Priego Moreno S*, Bailey R*, Campion N, Herron S, Gambus A. Polyubiquitylation drives replisome disassembly at the termination of DNA replication. Science, 2014 Oct 24;346(6208):477-81. doi: 10.1126/science.1253585.
- Moreno SP, Gambus A. Mechanisms of eukaryotic replisome disassembly. Biochem Soc Trans. 2020 Jun 30;48(3):823-836. doi: 10.1042/BST20190363
BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology
Techniques that will be undertaken during the project:
- Model systems: Xenopus laevis egg extract, in vitro proten work and human cell lines
- Recombinant protein production
- Protein biochemistry
- Electron microscopy, single molecule microscopy
- Cell biology – fluorescent microscopy, survival etc.
Contact: Dr Aga Gambus, University of Birmingham