[L29M] substitution in the interface of subunit-subunit interactions enhances Escherichia coli RecA protein properties important for its recombinogenic activity.

Genetic analysis of RecA protein chimeras and their ancestors, RecAEc (from Escherichia coli) and RecAPa (Pseudomonas aeruginosa) had allowed us to place these proteins with respect to their recombinogenic activities in the following order: RecAPa>RecAX21>RecAX20=RecAEc. While RecAX20 differs from RecAEc in five amino acid residues with two substitutions ([S25A] and [I26V]) at the interface of subunit interactions in the RecA polymer, RecAX20 and RecAX21 differ only by a single substitution [L29M] present at the interface. Here, we present an analysis of the biochemical properties considered important for the recombinogenic activity of all four RecA proteins. While RecAX20 was very similar to RecAEc by all activities analysed, RecAX21 differed from RecAEc in several respects. These differences included an increased affinity for double-stranded DNA, a more active displacement of SSB protein from single-stranded DNA (ssDNA), a decreased end-dependent RecAX21 protein dissociation from a presynaptic complex, and a greater accumulation of intermediate products relative to the final product in the strand-exchange reaction. RecAPa was more tolerant than RecAX21 only to the end-dependent RecA protein dissociation. In addition, RecAPa was more resistant to temperature and salt concentrations in its ability to form a presynaptic RecAPa::ATP::ssDNA filament. Calculations of conformational energy revealed that the [L29M] substitution in RecAX21 polymer caused an increase in its flexibility. This led us to conclude that even a small change in the flexibility of the RecA presynaptic complex could profoundly affect its recombinogenic properties.