Multicolor fluorescence imaging with Sapphire used to probe the structure of DNA recombinase

6 minute read


For the latest publications, promotions, and news on upcoming products sent weekly to your inbox


Got a question? Let us help! Describe the problem you’re having and one of our experts will reach out.

In a recent publication in Nature Communications, Caldwell et al used the multi-color fluorescence imaging capacity of the Azure Sapphire Biomolecular Imager to study the structure of DNA recombinase1.

The RecT family of recombinases contains over 1500 members. These enzymes bind to and catalyze the annealing of two complementary pieces of single-stranded DNA (ssDNA). No protein structures of RecT family members from bacteria or prophages have been solved. Some structures of RAD52, a potential human homolog of RecT, have been reported without DNA and with ssDNA, but not with two pieces of ssDNA which would represent an important intermediate of the annealing process.

Gel-shift assays imaged using fluorescent imaging on Azure Sapphire Biomolecular Imager
A portion of Figure 8a from Caldwell et al (2022). Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing. Gel-shift assays testing whether mutated forms of the LiRecT protein can bind to Cy3- and Cy5-labeled strands of ssDNA. For each mutant and the wild-type (WT) version of the protein, each strand was added individually (labeled in the figure as 3 for Cy3, 5 for Cy5) or sequentially (labeled 35). This portion of the figure shows that for some amino acids, double mutations interfered with DNA binding while single mutations did not. The gel was imaged on the Azure Sapphire Biomolecular Imager. Licensed under CC BY 4.0.

Solving the LiRecT Structure

In this new work, Caldwell et al have solved the structure of LiRecT, a member of the RecT family from the prophage Listeria innocua, bound to a DNA duplex intermediate. The 3.4Å structure shows that multiple LiRecT subunits form a helical filament with an external groove that binds an extended and “un-wound” DNA duplex. The structure confirms that there is some structural similarity between the RecT family and the human protein RAD52. Though there is a great deal of difference between the structures, the authors identified a common core of structural similarity in the DNA-binding groove, supporting the hypothesis that the enzymes share a common underlying mechanism of protein-mediated DNA annealing.

Designed for flexible choice in detection chemistry and samples

The new Sapphire FL brings precise quantitation of nucleic acids and proteins. As the first system on the market of its kind to allow user-interchangeable filter modules, the Sapphire FL offers a broad range of excitation and emission wavelengths.
Scientist changing optical modules on the new Azure Sapphire FL
Pick the modules that support your research. Changing the optical modules on the new Sapphire FL is simple and easy. The unique mechanism makes selecting lasers to match your dyes finally possible.

Can mutated proteins still bind to ssDNA?

To confirm the importance of specific amino acids that appeared to contact the DNA in their structure, the authors mutated 21 amino acid residues to see if changing them interfered with DNA binding. Gel-shift assays were used to see if the mutated proteins could still bind to ssDNA. RecT is known to bind weakly to individual strands of ssDNA and strongly to two complementary strands when they are added sequentially. Therefore, the authors used two complementary strands of ssDNA, one labeled with Cy3 and the other with Cy5, and conducted gel-shift assays in which the DNA strands were added individually or sequentially to the mutated enzymes. The results were imaged on the Sapphire, whose multi-color fluorescence capability allowed the researchers to detect the migration of each oligonucleotide on the same gel. The results generally supported the interactions suggested by the structure, though double mutations were often needed to completely disrupt DNA binding.

The work is an important contribution to understanding the mechanism of action of the RecT family and in what ways it resembles and differs from that of RAD52.

Have you published with an Azure instrument?

We’d love to read it! Email your publication to us and we’ll send you something for sharing.


  1. Caldwell BJ et al. Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing. Nat Commun. 2022;13(1):7855.

Let us show you just how easy getting good data can be. Fill out this form to be contacted by a life science expert today!

Check out recent blog posts

Shopping cart0
There are no products in the cart!
Continue shopping