RECA TESTS HOMOLOGY AT BOTH PAIRING AND STRAND EXCHANGE

RecA is a 38-kDa protein from Escherichia coil that polymerizes on sin gle-stranded DNA, forming a nucleoprotein filament that pairs with hom ologous duplex DNA and carries out strand exchange in vitro. To observ e the effects of mismatches on the kinetics of the RecA-catalyzed reco mbination reaction, we used assays based upon fluorescence energy tran sfer that can differentiate between the pairing and strand displacemen t phases, Oligonucleotide sequences that produced 2-14% mismatches in the heteroduplex product of strand exchange were tested, as well as co mpletely homologous and heterologous sequences, The equilibrium consta nt for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and a n increase in the rate of dissociation of the intermediates. In additi on, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches. The equilibrium constant for pairing and the rate constant for strand disp lacement both decreased 6-fold as the heterology increased to 14%, The se results suggest that discrimination of homology from heterology occ urs during both pairing and strand exchange.