G. Reddy et al., UPTAKE AND PROCESSING OF DUPLEX DNA BY RECA NUCLEOPROTEIN FILAMENTS -INSIGHTS PROVIDED BY A MIXED POPULATION OF DYNAMIC AND STATIC INTERMEDIATES, Biochemistry, 34(32), 1995, pp. 10194-10204
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.