Elucidation of the rotary mechanism of serine recombinases

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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.