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Roles of ubiquitylation during eukaryotic DNA replication

Principal Supervisor: Dr Aga Gambus

Secondary Supervisor(s): Dr Marco Saponaro

University of Registration: University of Birmingham

BBSRC Research Themes:

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Deadline: 4 January, 2024


Project Outline

DNA replication is a highly evolutionarily conserved process, however there is still much to discover about how eukaryotes regulate genome duplication. Perturbations in DNA replication lead to genomic instability, which drives aging but also cancer development. Over the last 10 years we have been elucidating how ubiquitylation drives the disassembly of replication machinery during DNA replication process and how dysregulation of this process can affect genome stability. Due to ours, and others, work we have now much better understanding of this fundamental process, but also a better appreciation that the posttranslational modification by ubiquitin plays a key regulatory function during the process of DNA replication. The aim of this project is therefore to explore wider role of ubiquitylation during DNA replication process: identify novel ubiquitylated substrates, understand the function of such ubiquitylation, explore whether it leads to removal of the ubiquitylated proteins by p97 segregase or whether it is the deubiquitylation that is essential to maintain the stability of the genome.

To deliver this aim we will use a combination of two model systems. The discovery science will be delivered in cell free Xenopus laevis egg extract system, while the impact on genome stability will be analysed in immortalized human cell lines. The student will have therefore the chance to gain a wide spectrum of expertise spanning different model systems and both biochemistry and cell biology techniques.

Techniques

  • recombinant protein expression and purification
  • protein biochemistry using Xenopus laevis egg extract (chromatin isolation, western blotting, immunoprecipitation, immunodepletion, radioactive nucleotide DNA incorporation)
  • cell biology (tissue culture, immunofluorescence, DNA fibre assay, FACS, biochemistry)
  • structural biology