Biochemical basis of hyper-recombinogenic activity of Pseudomonas aeruginosa RecA protein in Escherichia coli cells

The replacement of Escherichia coli recA gene (recA_Ec) with the Pseudomonas aeruginosa recA_Pa gene in Escherichia coli cells results in constitutive hyper-recombination (high frequency of recombination exchanges per unit length of DNA) in the absence of constitutive SOS response. To understand the biochemical basis of this unusual in vivo phenotype, we compared in vitro the recombination properties of RecA_Pa protein with those of RecA_Ec protein. Consistent with hyper-recombination activity, RecA_Pa protein appeared to be more proficient both in joint molecule formation, producing extensive DNA networks in strand exchange reaction, and in competition with single-stranded DNA binding (SSB) protein for single-stranded DNA (ssDNA) binding sites. The RecA_Pa protein showed in vitro a normal ability for cleavage of the E. coli LexA repressor (a basic step in SOS regulon derepression) both in the absence and in the presence (i.e. even under suboptimal conditions for RecA_Ec protein) of SSB protein. However, unlike other hyper-recombinogenic proteins, such as RecA441 and RecA730, RecA_Pa protein displaced insufficient SSB protein from ssDNA at low magnesium concentration to induce the SOS response constitutively. In searching for particular characteristics of RecA_Pa in comparison with RecA_Ec, RecA441 and RecA803 proteins, RecA_Pa showed unusually high abilities: to be resistant to the displacement by SSB protein from poly(dT); to stabilize a ternary complex RecA::ATP::ssDNA to high salt concentrations; and to be much more rapid in both the nucleation of double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-dependent ATP hydrolysis at pH 7.5. We hypothesized that the high affinity of RecA_Pa protein for ssDNA, and especially dsDNA, is the factor that directs the ternary complex to bind secondary DNA to initiate additional acts of recombination instead of to bind LexA repressor to induce constitutive SOS response.