BIOCHEMICAL BASIS OF HYPER-RECOMBINOGENIC ACTIVITY OF PSEUDOMONAS-AERUGINOSA RECA PROTEIN IN ESCHERICHIA-COLI-CELLS

The replacement of Escherichia coil recA gene (recA(Ec)) with the Pseu domonas aeruginosa recA(Pa) gene in Escherichia coil cells results in constitutive hyper-recombination (high frequency of recombination exch anges per unit length of DNA) in the absence of constitutive SOS respo nse. To understand the biochemical basis of this unusual in vivo pheno type, we compared in vitro the recombination properties of RecA(Pa) pr otein with those of RecA(Ec) protein. Consistent with hyper-recombinat ion 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. c oil 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-re combinogenic proteins, such as RecA441 and RecA730, RecA(Pa) protein d isplaced insufficient SSB protein from ssDNA at low magnesium concentr ation to induce the SOS response constitutively. In searching for part icular characteristics of RecA(Pa) in comparison with RecA(Ec), RecA44 1 and RecA803 proteins, RecA(Pa) showed unusually high abilities: to b e resistant to the displacement by SSB protein from poly(dT); to stabi lize a ternary complex RecA::ATP::ssDNA to high salt concentrations; a nd to be much move rapid in both the nucleation of double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-dependent ATP hydrolysis a t pH7.5. We hypothesized that the high affinity of RecA(Pa), protein f or ssDNA, and especially dsDNA, is the factor that directs the ternary complex to bind secondary DNA to initiate additional acts of recombin ation instead of to bind LexA repressor to induce constitutive SOS res ponse.