Elucidation of the rotary mechanism of serine recombinases

Every cell's genetic information is stored as sequences of basepairs in immensely long, thin double-helical DNA molecules. Cells contain enzymes called recombinases that can alter DNA sequences by cutting strands and rejoining the ends to new partners. The actions of these enzymes must be very precise, as they have the potential to cause damage to the DNA and concomitant loss of genetic information. The serine recombinases are one group of these "DNA cut and paste" enzymes, derived from bacteria and archaea. Molecules of the recombinase recognize and bind to specific DNA sequences called sites. Two recombinase-DNA complexes then come together, and the recombinase breaks the DNA strands at the centres of each site. An extraordinary process then takes place where one half of this large protein-plus-DNA complex rotates relative to the other half, swapping the positions of a pair of broken DNA ends. The swapped ends are then joined to their new partners, completing the editing of the DNA sequence. This rotation mechanism was controversial at first as no other enzymes do anything like it, and although we now have strong indirect evidence supporting rotation, we still know very little about how the serine recombinase enzyme achieves this remarkable feat. In this project, we will apply advanced methods that allow us to observe single enzyme-DNA complexes that are undergoing DNA rearrangement, so we can see rotation as it happens. We will make complexes that each contain two fluorescent dye molecules, placed so that the distance between them changes as rotation takes place. The amount of light absorbed by these dyes and the brightness of the light they give out as fluorescence will tell us how far apart they are. We can thus tell how fast the recombinase can rotate the DNA ends, whether it pauses at any times during rotation, and how the rotation process can be affected by experimental factors, changes in the DNA sequence, or mutations of the enzyme. Our previous studies have revealed that there is a smaller module at the heart of the recombinase-DNA complex which can bind DNA sites and bring them together just like the complete enzymes. We will use similar single-molecule experiments to test whether this very simple module can also cause rotation.
The manipulation of DNA molecules by serine recombinases has enormous potential in biotechnology, synthetic biology, and nanotechnology such as for the editing of specific faulty genes for disease treatment, or exploiting the intrinsic rotary mechanism in nanoscale molecular motors. Our project will provide new insights into the mechanisms of these enzymes that might lead to enhancement of their unique properties and development for new applications.

Our aim is to elucidate the properties of the subunit rotation mechanism proposed for the serine recombinases, a large group of enzymes that promote DNA site-specific recombination. Using Tn3 resolvase and Sin, two serine recombinases which have been extensively analysed in vitro and for which crystal structures are available, we will assemble reaction intermediate complexes that are labelled with donor and acceptor fluorophores so that changes in Förster Resonance Energy Transfer (FRET) can be observed. Single complexes undergoing recombination will be attached to the surface of microscope slides and observed by TIRF microscopy. The resulting data will be profiles of the fluorescence signals over several tens of seconds, and will allow us to measure key kinetic parameters including the rates of steps leading up to the "rotation-ready" intermediate and the rate of the subunit rotation step itself. In further experiments the effects of factors including temperature, pH, ionic strength, viscosity and specific recombinase mutations will be determined and compared to predictions of a structure-based model for subunit rotation. Semisynthetic supercoiled fluorophore-bearing plasmid substrate molecules will be prepared and tested in order to measure the effects of supercoiling on rotation and to compare the properties of activated recombinase mutants with the wild-type enzymes. The properties of a 4-helix synaptic complex proposed to comprise the core rotation module in all serine recombinases will be determined. We will also use single-molecule TIRF experiments to analyse the enigmatic mechanism of switchable (directional) recombination by the serine integrases, a special group of serine recombinases encoded by bacteriophages which are being applied in biotechnology and synthetic biology.

Who will benefit from this research?
(a) Biotech companies who seek to use serine recombinases as tools in biotechnology and gene therapy.
(b) Clinicians and those in the biomedical sector who are seeking new therapies for bacterial infection.
(c) Those developing DNA-based nanotechnology and computation systems for commercial purposes.
(d) Staff and students involved with the project who can acquire transferable skills.
(e) The general public in the UK and beyond.
How will they benefit?
(a) Biotech companies. Already, serine recombinases are being widely used for targeted gene integration, leading to industrially useful microbial strains and other transgenic organisms. Novel chimaeric serine recombinases can be reprogrammed to recombine at chosen sequences; longer-term aims are recombinase-mediated integration of therapeutic genes in vivo or ex vivo, and genetically modified therapeutic stem cells. Other potential applications include markerless genetic modification of crop plants, and therapeutic deletion of integrated retroviruses from the genomes of infected cells. A deeper understanding of the serine recombinase mechanism might lead to improvements in the functionality of recombinases being used for these purposes, and thus to commercial opportunities in biotechnology and medicine. The UK public will benefit from any healthcare advances as a result of this work, and also if we have a lead in this sector bringing economic wealth to the nation.
(b) Clinicians/biomedical sector. Serine recombinases have essential roles in bacterial DNA mobility and thus spread of drug resistance (for example, acquisition of the key drug resistance genes in MRSA and VRSA strains of Staphylococcus aureus). Understanding of the recombination mechanism might facilitate design of drugs to specifically target these antibiotic-resistant bacteria. In other bacteria, serine recombinases promote genetic rearrangements that result in alteration of cell surface proteins in order to evade attack by the immune system; inhibition of the recombinase by drugs might facilitate clearance of the infection. Our research might thus lead to commercial rewards and healthcare improvements.
(c) Nanotechnology industry. Subunit rotation, the subject of this project, might have direct applications in nanodevices. Also, the ability of serine recombinases to produce small DNA circles, catenanes, and topologically complex structures might be applicable in the construction of nanomachines. The potential use of serine recombinase systems for construction of DNA-based logic systems (in vitro or in cells) has already generated substantial interest. Commercialization of these systems is still in the future but the potential markets might be large.
(d) Staff/student transferable skills. The project clearly offers the opportunity to maintain and develop staff in the UK with skills relevant to the applications outlined above as well as other areas at the interfaces of chemistry, physics and biology. These skills are transferable to many areas of current commercial and medical interest including research on enzymes that act on DNA, such as topoisomerases and the DNA repair/homologous recombination machinery. Our project will also provide opportunities for training and inspiring students at postgraduate, undergraduate and school level.
(e) General public. The UK public will benefit directly from any commercialization pursued by UK-based companies as outlined above, which would create high-skill employment opportunities. They will also benefit from any downstream healthcare and technological improvements that result. This project lends itself very well to graphical representation, and throughout the term of the grant we will look for opportunities to explain and promote our work to the general public in schools, public exhibitions, forums such as Cafés Scientifiques, etc, in order to enhance their understanding and appreciation of Science.