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（Bacteriophage，简称噬菌体）是一种感染细菌的病毒。为了确保它们的长期生存，许多细菌将自己的DNA与宿主细胞的DNA结合在一起，这一过程被称为整合。然后，每当细胞的DNA被复制时，噬菌体DNA就被复制。整合是由一种称为位点特异性重组的机制引起的：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。在某个时候，噬菌体通过将其DNA从宿主基因组中切回（切除）来重新形成感染性病毒颗粒，这也是由整合酶促进的。方便的是，整合和切除系统可以在实验室中工作，而不需要细菌或细菌;我们可以使用含有整合酶识别和结合位点的纯化短DNA片段和纯化蛋白质。这些酶中的一个家族被称为丝氨酸整合酶，已经被证明是科学家们非常感兴趣的，因为它的高度“单向”反应;丝氨酸整合酶本身促进整合，但不切除，而当另一种称为RDF（重组方向因子）的噬菌体蛋白存在时，它的行为正好相反，促进切除，但不整合。这种行为意味着这些系统可以用作完全可控的双向开关。这些可以用于构建许多有用的生物设备，包括基于DNA的电子计算机模拟物，其中开关可以充当二进制数字（1或0）。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，在生物技术和医学中具有潜在的有用应用。为了最大限度地发挥丝氨酸整合酶的作用，我们应该确切地了解它们是如何工作的;但是它们的“单向开关”特性仍然是相当神秘的。本文提出的研究的主要目的是揭示丝氨酸整合酶在识别其DNA靶位点时形成的蛋白质+ DNA“复合物”的结构，并将它们聚集在一起进行DNA链断裂和重新连接。为此，我们将使用称为冷冻电子显微镜（cryo-EM）的最先进技术，该技术涉及单个蛋白质-DNA复合物的成像和这些组件的单个副本的分析，以获得三维结构。这一结构信息将首次揭示整合酶如何实现单向重组。然后，我们可以通过实验室的实验来测试我们关于该机制的新想法，在实验室中，我们修改蛋白质或DNA，看看对重组反应的影响。一旦我们知道这些细节，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，并可能考虑将丝氨酸整合酶模块纳入更大/更复杂的系统的方法。这项研究将在格拉斯哥大学的Laura Spagnolo博士的实验室进行，Laura Spagnolo博士是使用cryo-EM确定蛋白质-DNA复合物结构的专家，在位点特异性重组领域的专家Sean Coloms博士和马歇尔斯塔克教授的支持下，cryo-EM工作将在格拉斯哥大学的苏格兰高分子成像中心（SCMI）使用最新的cryo-EM设备进行。