Visualisation of the steps in the replisome assembly process. University of Birmingham in United Kingdom

Defects in the maintenance of genome integrity are the foremost drivers of cellular deterioration leading to neuropathology and cancer development in humans. 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 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 also use our unique expertise in peptidomimetic ligand design and discovery (e.g. Chem. Sci. 2024 DOI: 10.1039/d4sc02240h and ChemRxiv 2024 oi: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.

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. For informal inquiries contact Prof Aga Gambus at: [email protected]

Funding notes:

This PhD project is part of Midlands Integrative Biosciences Training Partnership (MIBTP) funded by BBSRC (Biotechnology and Biological Sciences Research Council). For more details of the MIBTP Doctoral Training Program please visit the website: https://www.birmingham.ac.uk/research/activity/mibtp UK and international students are welcomed to apply.

References:

Passaretti P, Cvetkovic MA, Costa A, Gambus A. Purification of replisomes from Xenopus laevis egg extract system for single particle cryo-EM analysis. STAR Protoc. 2024 Aug 8;5(3):103237. doi: 10.1016/j.xpro.2024.103237.

Cvetkovic MA, Passaretti P, Butryn A, Reynolds-Winczura A, Kingsley G, Skagia A, Fernandez-Cuesta C, Poovathumkadavil D, George R, Chauhan AS, Jhujh SS, Stewart GS, Gambus A, Costa A. The structural mechanism of dimeric DONSON in replicative helicase activation. Mol Cell. 2023 Nov 16;83(22):4017-4031.e9. doi: 10.1016/j.molcel.2023.09.029.

Epub 2023 Oct 10. PMID: 37820732 Kingsley G, Skagia A, Passaretti P, Fernandez-Cuesta C, Reynolds-Winczura A, Koscielniak K, Gambus A. DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation. Nucleic Acids Res. 2023 Aug 28:gkad694. doi: 10.1093/nar/gkad694.

Online ahead of print. PMID: 37638758 Cawood E.E., Baker E., Edwards T.A., Woolfson D.N.,* Karamanos T.K.,* and Wilson A.J., Understanding ß-strand mediated protein–protein interactions: tuning binding behaviour of intrinsically disordered sequences by backbone modification. Chem. Sci., 2024, 15, 10237 , DOI: 10.1039/d4sc02240h.