The structural basis for the mechanism of directional DNA recombination

DNA定向重组机制的结构基础

• 批准号: BB/W017571/1
• 负责人: Laura Spagnolo
• 金额: $ 66.4万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW017571%2F1
• 关键词: structural; basis; mechanism; directional; DNA

Bacteriophages ('phages') are viruses that infect bacteria. To ensure their long-term survival, many phages join their own DNA with that of their host cell, a process known as integration. The phage DNA then gets copied each time the cell's DNA is copied. Integration is brought about by a mechanism called site-specific recombination: an enzyme (integrase) promotes breaking and rejoining of DNA strands at two specific places (sites) in the phage and the host DNA, thus splicing the two together. At some point the phage re-forms infectious virus particles by cutting its DNA back out of the host genome (excision), and this is also promoted by the integrase. Conveniently, integration and excision systems can be made to work in the lab without needing phages or bacteria; we can use purified short pieces of DNA containing the sites that integrase recognizes and binds to, and purified proteins. One family of these enzymes called the serine integrases has proved to be of great interest to scientists because of its highly 'one-way' reactions; on its own a serine integrase promotes integration but not excision, whereas when another phage protein called RDF (recombination directionality factor) is present it behaves exactly the opposite, promoting excision but not integration. This behaviour means that these systems can be used as fully controllable two-way switches. These can be used for the construction of many sorts of useful biological devices including DNA-based analogues of electronic computers, where the switch can act as a binary digit (1 or 0). Combinations of switches can then allow living cells, such as bacteria or yeast, to process information and make simple decisions, with potentially useful applications in biotechnology and medicine.To maximize the usefulness of serine integrases we should understand exactly how they work; but their 'one-way switch' properties are still quite mysterious. The big aim of the research proposed here is to reveal the structures of the protein + DNA 'complexes' that serine integrases form when they recognize their DNA target sites and bring them together to perform DNA strand breaking and rejoining. To do this we will use a state-of-the-art technology called cryo-electron microscopy (cryo-EM), which involves the imaging of individual protein-DNA complexes and the analysis of individual copies of these assemblies to obtain a three-dimensional structure. This structural information will reveal for the first time how the integrase enzymes bring about one-way recombination. We can then test our new ideas about the mechanism by experiments in the lab, where we modify the proteins or the DNA and see what the effects are on the recombination reactions. Once we know these details, we can design new integrase-based systems for optimum performance in synthetic biological devices, and potentially think of ways to incorporate serine integrase modules into larger/more complex systems.This research will be carried out at the University of Glasgow in the laboratories of Dr. Laura Spagnolo, a specialist in determining the structures of protein-DNA complexes using cryo-EM, with support from Dr. Sean Colloms and Professor Marshall Stark who are experts in the field of site-specific recombination. The cryoEM work will be carried out at the Scottish Centre for Macromolecular Imaging (SCMI) at the University of Glasgow using the very latest cryo-EM equipment.

噬菌体（噬菌体）是感染细菌的病毒。为了确保它们的长期生存，许多噬菌体将自己的DNA与宿主细胞的DNA结合在一起，这一过程被称为整合。每次细胞的DNA被复制时，噬菌体DNA也会被复制。整合是通过一种称为位点特异性重组的机制实现的：一种酶（整合酶）促进噬菌体和宿主DNA中两个特定位置（位点）的DNA链断裂和重新连接，从而将两者拼接在一起。在某种程度上，噬菌体通过将其DNA从宿主基因组中切割出来（切除）来重组感染性病毒颗粒，这也由整合酶促进。方便的是，整合和切除系统可以在实验室工作，而不需要噬菌体或细菌；我们可以使用纯化的DNA短片段，包含整合酶识别和结合的位点，以及纯化的蛋白质。这些酶的一个家族被称为丝氨酸整合酶，由于其高度的“单向”反应而引起了科学家们的极大兴趣；丝氨酸整合酶本身促进整合但不促进切除，而当另一种称为RDF（重组方向性因子）的噬菌体蛋白存在时，它的行为恰恰相反，促进切除但不促进整合。这种行为意味着这些系统可以用作完全可控的双向开关。这些可用于构建多种有用的生物设备，包括基于dna的电子计算机类似物，其中开关可以作为二进制数字（1或0）。然后，开关的组合可以让活细胞，如细菌或酵母，处理信息并做出简单的决定，这在生物技术和医学上具有潜在的有用应用。为了最大限度地发挥丝氨酸集成的作用，我们应该确切地了解它们是如何工作的；但它们的“单向开关”特性仍然很神秘。这里提出的研究的主要目的是揭示蛋白质+ DNA“复合物”的结构，丝氨酸整合酶在识别DNA靶位点并将它们聚集在一起以执行DNA链断裂和重新连接时形成的复合物。为了做到这一点，我们将使用一种最先进的技术，称为冷冻电子显微镜（cryo-EM），它涉及到单个蛋白质- dna复合物的成像和分析这些组装的单个副本，以获得三维结构。这一结构信息将首次揭示整合酶如何导致单向重组。然后我们可以通过实验室的实验来测试我们关于机制的新想法，在那里我们修改蛋白质或DNA，看看对重组反应有什么影响。一旦我们了解了这些细节，我们就可以设计新的基于整合酶的系统，以在合成生物设备中获得最佳性能，并可能想到将丝氨酸整合酶模块整合到更大/更复杂的系统中的方法。这项研究将在格拉斯哥大学的Laura Spagnolo博士的实验室进行，Laura博士是使用冷冻电镜确定蛋白质- dna复合物结构的专家，并得到了位点特异性重组领域的专家Sean Colloms博士和Marshall Stark教授的支持。冷冻电镜工作将在格拉斯哥大学的苏格兰大分子成像中心（SCMI）进行，使用最新的冷冻电镜设备。