Resolution of the Three-stranded Recombination Intermediate Made by RecA Protein: An Essential Role of ATP Hydrolysis

Abstract

Previous work has shown that triplex DNA is an intermediate in homologous pairing and strand exchange promoted by RecA protein. Heterology at the proximal end of duplex DNA blocks strand exchange, but triplex joints form nonetheless at the homologous distal end. Experiments on the formation and processing of distal joints revealed that the yield of distal joints depends critically on the concentration of RecA-coated single strands and the adequacy of the ATP-regeneration system, and reflects a steady state. Distal joints reversibly formed and dissociated, as shown by several methods, including a chase with unlabeled duplex DNA. Controls excluded a contribution of exonucleolytic nibbling to the formation of distal joints and the stability of the deproteinized product. RecA protein was bound preferentially by putative triplex sites both in isolated proximal and distal joints. These high affinity sites disappeared from proximal joints as strand exchange progressed, and disappeared from distal joints as the joints dissociated. Dissociation of distal joints under all conditions, however, was completely arrested by the addition of ATPγS. Distal triplex joints can be as long as six kilobases. The observed inhibition of the dissociation of such long non-productive triplex intermediates by ATPγS leads us to propose that an essential role of ATP hydrolysis in RecA recombinational exchanges may be to ensure that no potentially troublesome triplex DNA remains in the cell.