Single molecule resolution imaging of stressed DNA - the next paradigm shift in cancer therapy design?

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Background: Changes in genomic stability, DNA replication, DNA damage and repair underpin carcinogenesis. Over the last 15 years the Bryant lab has pioneered targeting the DNA damage response (DDR) in cancer. This has caused a shift in precision medicine (e.g. PARP inhibitors), whilst drugs that alter replication (e.g. topoisomerase inhibitors) remain the cornerstone of cancer therapy. Despite this, the topology of DNA during cancer cell replication and under therapy is poorly understood because we lack tools to quantitatively determine the topological state of DNA. Here we partner with the Pyne lab to combine cell biology and high-resolution single-molecule imaging to understand how the topological landscape of DNA during replication affects DNA processing to understand and identify novel opportunities for therapeutic intervention. Objectives:1. Quantify the topology of DNA intermediates formed during cancer cell replication2. Determine how oncogene overexpression alters the topology of DNA intermediates3. Establish how the topology of DNA affects the activity of chemotherapeutic agents and correlate this with cellular survival, replication kinetics and the DNA damage responseNovelty: This project applies our cutting-edge single-molecule Atomic Force Microscopy (AFM) techniques, capable of visualising the double helix of individual DNA molecules, to determine the structure of previously undefined replication/repair intermediates. AFM allows us to explore for the first time how oncogene and therapy induced replicative stress affects the structure and conformation of DNA intermediates. Given the emergence of replication and DDR inhibitors as therapeutic agents, and the increasing awareness of the biophysical forces that act on DNA to influence cell behaviour, this project is perfectly positioned to progress our understanding of how DNA topology contributes to cancer progression and therapy response.Experimental Approach:You will receive training in high-resolution single-molecule imaging, cell biology and oncology. You will use high-resolution AFM to visualise individual DNA intermediates with double helical resolution and develop our quantitative image analysis tools to explicitly determine the topology of intermediate structures. You will use our inducible cell lines to overexpress oncogenes such as MYC, RAS and cyclin E; and a range of chemotherapeutic agents to induce replicative stress. You will correlate AFM findings with cellular DNA damage response assays including survival, molecular fibres (replication speed/stalling), COMET assays (DNA breaks) and immunofluorescence (repair foci and R-loops). We collaborate internationally with academia and industry across the sciences and engineering, with downstream impact in therapeutic development. You will join a collaborative, supportive research community at Sheffield, with world-leading single-molecule and nucleic acid research centres.