Visualisation of the steps in the replisome assembly process

Secondary Supervisor(s): Professor Andrew Wilson

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Structural Biology)

Project Outline

Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. The replication, repair and epigenetic regulation of the human genome are three of nine interrelated pathways involved in ageing. Defects in the maintenance of genome integrity are the foremost drivers of cellular deterioration leading to neuropathology and cancer development in humans. Specifically, the issue of how vertebrates, including humans, select and activate origins of replication are not resolved conclusively and are of importance; areas of the genome with fewer replication origins are more prone to genomic instability whilst mutations in factors involved in origin activation lead to rare human diseases.

Over the last 15 years, biochemical and structural protein work based on yeast proteins, has unveiled a much better understanding of how the replication machinery is assembled during origin activation, which, has transformed our understanding of DNA replication initiation in this simple eukaryote. However, we have shown recently that the mechanism of replication machinery assembly in higher eukaryotes, including in humans, differs significantly. Using biochemical approaches in cell free Xenopus laevis egg extract we have identified DONSON as an essential replication initiation factor in vertebrates (Nucleic Acids Research, 2023 doi: 10.1093/nar/gkad694). Moreover, we have devised a method to isolate replication machinery complexes assembled at replicating chromatin in Xenopus egg extract, so that we can visualise them using Cryo-electron microscopy (Cryo-EM) (Star Protocols 2024, doi: 10.1016/j.xpro.2024.103237). By doing this, we have provided evidence of the structural importance of DONSON during activation of the replication machinery (Mol Cell 2023, doi: 10.1016/j.molcel.2023.09.029). This new revolutionary approach to visualise the structural aspects of DNA replication complexes now allows us to tackle other steps in this process and deliver equally impactful and transformative discoveries.

The aim of this project is to utilise our novel approach to visualise steps in replication machinery assembly, upstream and downstream of DONSON action. We have established that we can use a series of inhibitors to enrich for complexes inhibited at particular stages of replication initiation. We will also remove essential initiation factors by immunodepletion to discover the structure of the machinery frozen at the step in which they are required. Once novel structures are determined they will be validated through biochemical approaches. We will create recombinant proteins with mutated residues important for particular interactions. Such proteins will be purified and tested in the cell free Xenopus egg extract to determine if they are functional and able to support DNA replication, analogously as we have done to validate the structure of CMG-DONSON (Mol Cell 2023). We will also use our unique expertise in peptidomimetic ligand design and discovery (e.g. Chem. Sci. 2024 doi: 10.1039/d4sc02240h and ChemRxiv 2024 doi:10.26434/chemrxiv-2024-299w9) alongside the obtained structures to design short peptidomimetics to disrupt essential protein-protein interactions within the complex to inhibit this particular stage of replication initiation. We will determine the consequences of such inhibition for replication in egg extract and use the generated inhibitory peptidomimetics iteratively – to freeze in time earlier stages of replication initiation complexes.

We also have the expertise to validate our findings in human immortalised cell lines, where we can rapidly degrade endogenous protein using an auxin inducible degron (Nat Comms 2023) and express mutants for functional testing.

This project will therefore combine biochemistry in Xenopus laevis egg extract, structural biology to deliver the cryoEM structures, chemistry to synthesise peptidomimetics and cell biology to follow on findings in human cells.