Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson–Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8–20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer–monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics.

RecA plays a central role in DNA repair and is a main actor involved in recombination and activation of the SOS response. It is also used in the context of biotechnological applications in recombinase polymerase isothermal amplification (RPA). In this work, we studied the biological properties of seven RecA variants, in particular their recombinogenic activity and their ability to induce the SOS response, to better understand the structure–function relationship of RecA and the effect of combined mutations. We also investigated the biochemical properties of RecA variants that may be useful for the development of biotechnological applications. We showed that Dickeya dadantii RecA (DdRecA) had an optimum strand exchange activity at 30 °C and in the presence of a dNTP mixture that inhibited Escherichia coli RecA (EcRecA). The differences between the CTD and C-tail of the EcRecA and DdRecA domains could explain the altered behaviour of DdRecA. D. radiodurans RecA (DrRecA) was unable to perform recombination and activation of the SOS response in an E. coli context, probably due to its inability to interact with E. coli recombination accessory proteins and SOS LexA repressor. DrRecA strand exchange activity was totally inhibited in the presence of chloride ions but worked well in acetate buffer. The overproduction of Pseudomonas aeruginosa RecA (PaRecA) in an E. coli context was responsible for a higher SOS response and defects in cellular growth. PaRecA was less inhibited by the dNTP mixture than EcRecA. Finally, the study of three variants, namely, EcPa, EcRecAV1 and EcRecAV2, that contained a combination of mutations that, taken independently, are described as improving recombination, led us to raise new hypotheses on the structure–function relationship and on the monomer–monomer interactions that perturb the activity of the protein as a whole.