Being seen on Holliday - a universal marker for comparing DNA repair by homologous recombination in multiple forms of life

该项目将开发一种通过同源重组(HR)识别DNA修复的新方法，并将其部署在细菌、人类、古菌和酵母中。HR由多个相关的亚通路组成，它们克服了复制DNA的中断，在癌症生物学、减数分裂和原核生物的遗传流动中起着至关重要的作用。所有生物体中的HR通路都有共同的初始事件，即检测DNA链断裂，将其切除，并侵入同源的未断裂DNA。DNA断裂和切除可以通过各种方法检测到，表明HR正在进行中。然后，HR可以分成多个子通路。定义它们很复杂，部分原因是它们的检测方法是间接的。我们的目标是通过DNA复制直接检测到一个主要的途径结果-长道HR，这种DNA复制形成了称为Holliday Junctions的特殊DNA结构。这将有助于准确了解是什么因素指导和控制HR途径的选择。我们将利用RUSA蛋白来可视化Holliday连接。这种小的(14 KDa)蛋白质识别Holliday连接具有高度的特异性，并将它们切割以恢复双链DNA。RUSA最初是在细菌中发现的，后来被改造成核定位信号(NLS)，并与绿色荧光蛋白(GFP)“标签”融合，用于庞贝葡萄球菌。在这个博士项目中，我们将使用具有催化活性的细菌RUSA酶和一个突变版本(RusAD70N)，该突变版本与Holliday连接结合，但无法切割它们，因此允许Holliday连接持续存在。这将提供可视化的手段，以响应DNA断裂的Holliday连接的形成。首先，我们将在大肠杆菌、酿酒酵母、人骨肉瘤细胞(U2OS)和古生代Haloferax Volcanii中检测到带有和不带有NLS的GFP-RUSA和GFP-RusAD70N蛋白(根据细胞类型)。在不同基因毒性物质诱导的HR过程中，将通过共聚焦显微镜(Bolt Lab)对这些蛋白质进行比较，以可视化Holliday连接的形成，预测GFP-RUSA焦点应该增加以响应基因毒性应激。博尔特-艾勒斯-格雷实验室经常使用这些细胞类型和HR诱导治疗。建立GFP-RUSA/RusAD70N成像将促进该项目的下一阶段，以确定每种细胞类型中将HR转向Holliday连接形成的因素。我们将使用遗传学来删除或激活每种细胞类型中编码我们的候选蛋白质的基因。这是通过在人类U2OS细胞中使用CRISPR/Cas9编辑以及在酿酒酵母、细菌和古生菌中进行重组工程实现的--这些技术已经在Bolt-Aller-Gray实验室中到位。然后，将通过在人类U2OS细胞中使用Cas9-RT‘Prime’编辑和在大肠杆菌中使用‘Retron编辑’引入精确定义的点突变来仔细检查任何在这方面被识别为有趣的基因。后一种方法还没有在酵母菌或古生菌中进行测试，但我们预计它们也可能成为这个博士项目的一部分。这些研究旨在发现新的调节HR亚通路的分子机制，可在体外进一步研究。

The project will develop a new approach to identifying DNA repair by homologous recombination (HR), and deploy it in bacteria, humans, archaea, and yeast. HR comprises multiple related sub-pathways that overcome breaks in replicating DNA, with crucial roles in cancer biology, meiosis, and genetic flux in prokaryotes. HR pathways in all organisms share common initial events that detect DNA strand breaks, resect them, and invade into homologous unbroken DNA. DNA breaks and resection can be detected by various means showing HR is underway. HR can then diverge into multiple sub-pathways. Defining these is complex in part because of indirect methods for their detection. We aim to directly detect one major pathway outcome - long tract HR by DNA replication that forms specialised DNA structures called Holliday junctions. This will help to understand exactly what factors direct and control HR pathway choice.We will utilize the RusA protein to visualise Holliday junctions. This small (14 kDa) protein recognizes Holliday junctions with high specificity and cuts them to restore duplex DNA. RusA was originally identified in bacteria and has since been engineered with a nuclear localisation signal (NLS) and fused to a green-fluorescent protein (GFP) 'tag' for use in S. pombe yeast. In this PhD project we will use the catalytically active bacterial RusA enzyme alongside a mutant version (RusAD70N) that binds to Holliday junctions but is unable to cut them, therefore allowing Holliday junctions to persist. This will provide the means to visualise Holliday junction formation in response to DNA breaks. First, we will detectably produce GFP-RusA and GFP-RusAD70N proteins with and without an NLS (as appropriate for cell type) in E. coli, S. cerevisiae, human bone osteosarcoma cells (U2OS) and the archaeon Haloferax volcanii. The proteins will be compared for visualising Holliday junction formation, by confocal microscopy (Bolt lab), during HR induced by various genotoxic agents, predicting that GFP-RusA foci should be increased in response to genotoxic stress. Each of these cell types and HR-inducing treatments are frequently used in the Bolt-Allers-Gray labs. Establishment of GFP-RusA/RusAD70N imaging will facilitate the next stage of the project, to define factors in each cell type that divert HR towards Holliday junction formation. We will use genetics to delete or activate genes in each cell type that encode our candidate proteins. This is achieved using CRISPR/Cas9 editing in the human U2OS cells and by recombineering in S. cerevisiae, bacteria, and archaea - techniques already in place in the Bolt-Allers-Gray labs. Any genes identified as interesting in this context will then be scrutinised by introducing precisely defined point mutations using Cas9-RT 'Prime' editing in human U2OS cells, and 'Retron editing' in E. coli. These latter methods are not yet tested in yeasts or archaea, but we expect that they may also form part of this PhD project. These investigations aim to identify new molecular mechanism in regulation of HR sub-pathways, that can be developed in further work in vitro.