Homologous pairing in genetic recombination. The pairing reaction catalyzed by Escherichia coli recA protein

Abstract

Purified recA protein, which is essential for genetic recombination of Escherichia coli, catalyzed ATP-dependent homologous pairing of double-stranded DNA and single-stranded fragments to form D-loops. When the double-stranded DNA was nicked circular DNA (form II) or linear DNA (form III), the reaction proceeded nearly linearly during 30 min of incubation at 37 degrees C. When the double-stranded DNA was superhelical (form I), anomalous kinetics was observed. This anomaly was suppressed by the addition of spermidine without affecting the final yield of D-loops. The formation of D-loops required stoichiometric amounts of recA protein, which were proportional to the concentration of single-stranded DNA but which were not affected by the concentration of double-stranded DNA. With form II or III DNA as the recipient for the formation of D-loops, the rate of the reaction was greatest when there was one monomer of recA protein/2-3 nucleotide residues of single-stranded DNA; larger amounts of single-stranded DNA inhibited the reaction. The formation of D-loops was half inhibited by 30 mM NaCl and by 0.6 mM ADP, one of the products of the reaction. The thermal stability of D-loops made by recA protein was the same as that of D-loops made by annealing. In addition to pairing linear single strands with duplex DNA, recA protein made joint molecules from single-stranded circular DNA and homologous form II or III DNA. According to these and previous observations (Cunningham, R. P., DasGupta, C., Shibata, T., and Radding, C. M. (1980) Cell 20, 223-235), rcA protein will stably pair two molecules of DNA if one of them is single-stranded or partially single-stranded and if either molecule has a free end.