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。为了确保其长期生存，许多噬菌体将自己的DNA与宿主细胞的DNA一起加入，这一过程称为整合。每次复制细胞的DNA时，噬菌体DNA都会复制。积分是由一种称为位点特异性重组的机制引起的：酶（整合酶）促进了在噬菌体和宿主DNA中两个特定位置（位置）的DNA链的破裂和重新加入，从而将两者拼凑在一起。在某个时候，噬菌体通过将其DNA从宿主基因组中切出（切除）来重新形成感染性病毒颗粒，这也由整合酶促进。方便地，可以使整合和切除系统在实验室工作，而无需噬菌体或细菌。我们可以使用纯化的短片DNA，其中包含积分酶识别并结合并结合并纯化的蛋白质的位点。这些酶中的一个家族称为丝氨酸综合酶，由于科学家的“单向”反应高度非常重要。丝氨酸积分酶自身促进了整合而不是切除，而当存在另一个称为rdf的噬菌体蛋白（重组方向性因子）时，它的行为恰恰相反，促进了切除，而不是整合。这种行为意味着这些系统可以用作完全可控制的双向开关。这些可用于构建多种有用的生物学设备，包括电子计算机的基于DNA的类似物，该开关可以用作二进制数字（1或0）。然后，开关的组合可以允许活细胞（例如细菌或酵母菌）处理信息并做出简单的决定，并在生物技术和药物中有可能有用的应用。为了最大程度地利用丝氨酸积分的有用性，我们应该确切地了解它们的工作方式；但是他们的“单向开关”属性仍然很神秘。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，即丝氨酸识别其DNA靶位点并将它们聚集在一起以进行DNA链破裂和重新加入。为此，我们将使用一种称为Cryo-Electron显微镜（Cryo-EM）的最先进的技术，该技术涉及单个蛋白DNA复合物的成像以及对这些组件的各个副本的分析以获得三维结构。这些结构信息将首次揭示整合酶如何带来单向重组。然后，我们可以通过实验中的实验测试有关机制的新想法，在该实验中，我们修改蛋白质或DNA，并查看对重组反应的影响。一旦知道了这些细节，我们就可以设计新的基于集成酶的系统，以在合成生物学设备中的最佳性能，并可能考虑将丝氨酸整合酶模块纳入较大/更复杂的系统中。这项研究将在格拉斯哥的Glasgow实验室进行，该研究由Laura Spagnolo的实验室中的实验室进行。斯塔克（Stark）是特定地点重组领域的专家。冷冻工作将在格拉斯哥大学的苏格兰大分子成像中心（SCMI）使用最新的Cryo-EM设备进行。