Award details

The structural basis for the mechanism of directional DNA recombination

Reference	BB/W017571/1
Principal Investigator / Supervisor	Dr Laura Spagnolo
Co-Investigators / Co-Supervisors	Dr Sean Colloms, Professor Marshall Stark
Institution	University of Glasgow
Department	College of Medical, Veterinary, Life Sci
Funding type	Research
Value (£)	521,107
Status	Current
Type	Research Grant
Start date	01/04/2023
End date	31/03/2026
Duration	36 months

Abstract

Serine integrases, encoded by bacteriophages for integration and excision of their genomes to/from their host cell's DNA, are an important group of recombinases which have attracted much attention because of their ability to promote highly directional site-specific recombination reactions. Their ability to recombine DNA in both in vitro and in vivo systems, as well as logging biological cues and executing logic functions, made serine integrases excellent candidates to be applied in biotechnology and synthetic biology. This project aims to establish the mechanistic features that determine recombination directionality in integrases. To achieve this, we will adopt a two-pronged strategy: 1. We will use cryoEM to directly determine the structures of serine integrase complexes with the individual recombination sites (attP, attB, and attL/R), and synaptic complexes formed by pairs of recombination sites. These structures will include those made using mutant versions of the integrase protein designed to promote the formation of intermediates in the reaction pathway. We will also determine equivalent structures formed by integrases and their sites with the RDF. These structures will reveal the positions and interactions of the integrase domains (including the coiled-coil domains), and the positions of the RDF subunits when they are present. 2. We will characterize the intermediate complexes in serine integrase-mediated recombination using genetic (mutation) and biochemical (including gel electrophoretic and chromatographic) methods, to reveal how these complexes are affected by mutations of the protein or att site DNA, and what steps in the reaction pathway are crucial for directionality. The cryoEM, genetic and biochemical experiments will be mutually informative, and will be essential in future work aimed at refining the current mathematical model.

Summary

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.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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