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.
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.
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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.
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SOURCE
- 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.
