Project Outline

Housed within the nucleus of the cell, DNA replication is the fundamental process of cell division and occurs within the majority of the trillions of cells we have in the body. Understanding how it is regulated in healthy cells is of the utmost importance, so that we can then exploit our findings and tackle the many diseases associated with defective DNA replication, including ageing and cancer.

In eukaryotic cells, with large genomes, DNA replication begins at thousands of sites along the chromatin, named origins of replication. Selection and activation of these origins is a multi-stage process, with each step occurring in different stages of the cell cycle. Origins are first selected by the Origin Recognition Complex (ORC) in late mitosis, are licensed by loading of the MCM2-7 helicase complex in G1 phase and then activated in S-phase (Costa and Diffley, 2022. Ann Rev Biochem). The focus of this project is to characterise the first step of origin selection by the ORC complex in human cells. Although generally accepted that this occurs in late mitosis, very little is currently known as to how this is coordinated, especially in the context of other late-mitotic events. One such, very important, late-mitotic event is the re-assembly of the nuclear envelope around the segregated chromosomes. Notably, both ORC binding and nuclear envelope reassembly, are thought to occur in late anaphase/early telophase (Diffley et al, 1994. Cell; Guttinger et al, 2009. Nat Reviews).

The aim of this project is to explore the connection between the selection of replication origins by the ORC complex and nuclear envelope reassembly following exit from mitosis. For this, we will work primarily with immortalised human cell lines and use a range of cell biology, microscopy and biochemical techniques to characterise the binding of these factors to chromatin and begin to explore whether they impact one-another.

This project would suit a PhD student candidate who has an interest in studying and characterising fundamental biological processes in cells, and a passion for learning new molecular biology and biochemical techniques.

Techniques

• Cell biology (tissue culture, flow cytometry)
• Microscopy
• Molecular biology (protein expression, molecular cloning)
• Biochemistry (western blotting, chromatography)
• Mass spectrometry