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

背景：基因组稳定性、DNA复制、DNA损伤和修复的变化是癌症发生的基础。在过去的15年里，布莱恩特实验室率先针对癌症中的DNA损伤反应（DDR）进行研究。这导致了精准医学（如PARP抑制剂）的转变，而改变复制的药物（如拓扑异构酶抑制剂）仍然是癌症治疗的基石。尽管如此，由于我们缺乏定量确定DNA拓扑状态的工具，因此对癌细胞复制和治疗过程中的DNA拓扑结构知之甚少。在这里，我们与Pyne实验室合作，将细胞生物学和高分辨率单分子成像相结合，以了解复制过程中DNA的拓扑景观如何影响DNA加工，从而了解和确定治疗干预的新机会。目的:1。量化癌细胞复制过程中形成的DNA中间体的拓扑结构2。确定癌基因过表达如何改变DNA中间体的拓扑结构3。建立DNA拓扑结构如何影响化疗药物的活性，并将其与细胞存活、复制动力学和DNA损伤反应联系起来。高度：该项目应用我们尖端的单分子原子力显微镜（AFM）技术，能够可视化单个DNA分子的双螺旋结构，以确定以前未定义的复制/修复中间体的结构。AFM允许我们首次探索癌基因和治疗诱导的复制应激如何影响DNA中间体的结构和构象。鉴于复制和DDR抑制剂作为治疗剂的出现，以及对作用于DNA以影响细胞行为的生物物理力的日益认识，该项目完美地定位于推进我们对DNA拓扑结构如何促进癌症进展和治疗反应的理解。实验方法：您将接受高分辨率单分子成像，细胞生物学和肿瘤学方面的培训。您将使用高分辨率AFM以双螺旋分辨率可视化单个DNA中间体，并开发我们的定量图像分析工具来明确确定中间体结构的拓扑结构。您将使用我们的诱导细胞系过度表达癌基因，如MYC、RAS和细胞周期蛋白E；以及一系列化疗药物来诱导复制应激。您将把AFM结果与细胞DNA损伤反应分析联系起来，包括存活、分子纤维（复制速度/延迟）、COMET分析（DNA断裂）和免疫荧光（修复焦点和r环）。我们在科学和工程领域与学术界和工业界进行国际合作，对治疗开发产生下游影响。您将加入谢菲尔德的协作性，支持性研究社区，拥有世界领先的单分子和核酸研究中心。

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.