UPTAKE AND PROCESSING OF DUPLEX DNA BY RECA NUCLEOPROTEIN FILAMENTS -INSIGHTS PROVIDED BY A MIXED POPULATION OF DYNAMIC AND STATIC INTERMEDIATES

In the polarized strand exchange that is promoted by Escherichia coli RecA protein, when the initiating end of a duplex DNA molecule is bloc ked by heterology, the homologous distal end nonetheless forms a joint with single-stranded DNA, but strand exchange in that joint cannot be completed because the strand that would otherwise be displaced lacks a free 5' end. Instead, 2/3 to 3/4 of such distal joints cyclically fo rm and dissociate. Dissociation requires the hydrolysis of ATP (Burnet t et al., 1994). Observations on DNase protection revealed that consis tent with their dynamic nature, these joints were heterogeneous in len gth, extending from the labeled distal end of the duplex up to 600 bas e pairs within the homologous region. Switching of base pairs was unde tectable in this fraction of distal joints. However, the other 1/3 to 1/4 of distal joints, which did not cycle, were as long as the entire homologous region (6 kb), and underwent complete switching of base pai rs. The formation of these static joints occurred at a rate in excess of 100 bp per second, without requiring hydrolysis of ATP. These and e arlier observations suggest that the RecA filament containing single-s tranded DNA rapidly incorporates duplex DNA into a coaxial three-stran ded helix by a passive process, whereas additional energy is required to convert the three-stranded intermediate into products or back into substrates, both of which involve the unwinding of many turns of three -stranded helix.