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，古细菌和酵母中。HR包括多个相关的子途径，这些子途径克服了复制DNA中的断裂，在原核生物的癌症生物学、减数分裂和遗传通量中起着至关重要的作用。所有生物体中的HR途径都有共同的初始事件，即检测DNA链断裂，切除它们，并侵入同源未断裂的DNA。DNA断裂和切除可以通过各种手段检测到，表明HR正在进行中。然后，HR可以分为多个子路径。定义这些是复杂的，部分原因是它们的检测方法是间接的。我们的目标是直接检测一个主要的途径结果-长道HR通过DNA复制形成专门的DNA结构，称为霍利迪连接。这将有助于准确地了解哪些因素指导和控制HR途径的选择。我们将利用RusA蛋白可视化霍利迪连接。这种小的（14 kDa）蛋白质识别霍利迪连接具有高特异性，并切割它们以恢复双链DNA。RusA最初是在细菌中发现的，后来被设计成具有核定位信号（NLS），并与绿色荧光蛋白（GFP）“标签”融合，用于S。粟酒酵母在这个博士项目中，我们将使用催化活性的细菌RusA酶以及一种突变体（RusAD 70 N），该突变体与霍利迪连接点结合，但无法切割它们，因此允许霍利迪连接点持续存在。这将提供可视化霍利迪连接形成响应DNA断裂的手段。首先，我们将在E. coli、S.酿酒酵母、人骨肉瘤细胞（U2 OS）和古菌Haloferax volcano ii。在由各种遗传毒性剂诱导的HR期间，通过共聚焦显微镜（Bolt实验室）比较蛋白质以可视化Holliday连接形成，预测GFP-RusA病灶应响应于遗传毒性应激而增加。这些细胞类型和HR诱导治疗中的每一种都经常在Bolt-Allers-Gray实验室中使用。GFP-RusA/RusAD 70 N成像的建立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候选蛋白质的基因。这是通过在人U2 OS细胞中使用CRISPR/Cas9编辑和在S.酿酒酵母，细菌和古细菌-技术已经在博尔特-阿勒斯-格雷实验室。然后将通过在人U2 OS细胞中使用Cas9-RT“Prime”编辑和在E.杆菌后一种方法尚未在酵母或古菌中进行测试，但我们预计它们也可能成为本博士项目的一部分。这些研究的目的是确定新的分子机制，在调节HR子途径，可以在进一步的工作在体外开发。

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.