Stable three-stranded DNA made by RecA protein.

Abstract

When RecA protein, in the form of a nucleoprotein filament containing circular single-stranded DNA (plus strand only), reacts with homologous linear duplex DNA, a directional transfer ensues of a strand from the duplex DNA to the nucleoprotein lament, resulting in the displacement of the linear plus strand in the 5' to 3' direction. The initial homologous synapsis, however, can occur at either end of the duplex DNA, or anywhere in between, and when homology is restricted to different regions of the duplex DNA,, the joint molecules that form in each region show striking differences in stability upon deproteinization: distal joints > proximal joints >> medial joints. In the deproteinized distal joints, which are thermostable, 2000 nucleotide residues of the circular plus strand are resistant to P1 nuclease; both strands of the original duplex DNA remain resistant to P1 nuclease, and the potentially displaceable linear plus strand, which has a 3' homologous end, remains resutant to Escherichia coli exonuclease I. These observations suggest that RecA protein promotes homologous pairing and strand exchange via long three-stranded DNA intermediates and, moreover, that, once formed, such triplex structures in natural DNA are stable even when RecA protein has been removed.A model system that has proven useful in exploring the nature of recombination promoted by RecA protein consists of circular single-stranded DNA and duplex DNA, the latter usually in linear form (1,2). Studies ofthis model system have revealed the overall course of the reaction and some mechanistic aspects. The reaction has three major phases: (i) presynapsis, in which RecA protein polymerizes on singlestranded DNA to form a right-handed helical nucleoprotein filament; (ii) synapsis, during which naked duplex DNA first binds nonspecifically and weakly to sites on the nucleoprotein filament and then comes into homologous alignment; and (iii) strand exchange, which produces a new heteroduplex molecule and a displaced linear single strand.Strand exchange itself is directional, 5' to 3' with respect to the circular plus strand in the RecA filament or its displaced linear homolog, which defines a proximal and a distal end in the duplex DNA ( Fig. 1; refs. 1 and 2). Many observations have been reported that show, however, that the initial pairing event that produces a joint molecule is not polarized but rather can occur at the proximal 5' end of the plus strand, the distal 3' end, or in the middle of a linear duplex molecule (Fig. 1;. On the other hand, Konforti and Davis (6,7), studying the pairing of linear single strands with superhelical DNA as assayed by gel electrophoresis, found that when homology was limited to the 5' end of a linear plus strand, joints were scarcely detectable. Hsieh et al. (8) found that when homology was limited to very short sequences at the ends of duplex DNA, only distal joints, as defined above, were detectable by gel electrophoresis. Since strand exchange, which proceeds in the 5' to 3' direction, was previously ob...

Stable synapsis of homologous DNA molecules mediated by the Escherichia coli RecA protein involves local exchange of DNA strands.

“…The experiments in this discussion employed short ssDNA with homology restricted to the middle of the linear target dsDNA, whereas Hsieh and Rao and their coworkers used a long circular ssDNA and a linear dsDNA in which the homology (ranging from 38-56 bp in length in one study to a few kilobases in another) is restricted to the 5' end of the recipient strand of the dsDNA. When the homology was restricted to the middle or the other end of the dsDNA, the stable, protein-free, three-stranded structure was not detected (Rao et al 1991), in agreement with the results reported here. If the putative triplex DNA involving non-WatsonCrick base-pairing is one of the stable intermediates of DNA synapsis as proposed (Hsieh et al 1990;Rao et al 1991), it will be interesting to investigate why the efficient formation and/or stability of this structure require a particular end of DNA, as this has been unprecedented up to now in canonical DNA triple helices.…”

“…These results are not only consistent with the DMS-reactivity data reported here but also argue against the view that the process of homology recognition involves an intermediate in which N-7 of G plays a critical role. The results in this discussion are not necessarily at odds with those reported by Hsieh et al (1990) and Rao et al (1991), who found a stable DNA structure after the removal of RecA in which all three strands appear to be somehow juxtaposed. The apparently different experimental outcome may be simply due to differences in experimental conditions, most notably the combination of DNA substrates used.…”

“…One may envision some kind of triplex DNA in which the ssDNA is bonded to the intact dsDNA by, for instance, non-Watson-Crick base-pairing (Howard-Flanders et al 1984); exchange of DNA strands occurs after homology recognition. In favor of this model, Hsieh et al (1990) and Rao et al (1991) reported that RecA can mediate formation of a proteinfree, stable DNA structure whose properties were proposed to be consistent with the stable triplex DNA with additional base-pairings.…”

The duplex DNA is very underwound in the three‐stranded RecA protein‐mediated synaptic complex

“…Second, thymines within the synaptic complex, but close to the reactive flank, are hyper-reactive in all three strands. This asymmetry in modification of two flanking regions probably reflects the polarity of presynaptic filament assembly and subsequent strand exchange (Konforti & Davis 1990Dutreix et al 1991).…”

Marking of specific sequences in double-stranded DNA molecules—SNP detection and direct observation

“…RecA protein is capable of directly recognizing specific base sequences in double-stranded DNA molecules and is known to form a triple-stranded structure with a complementary oligonucleotide, but the triple-stranded structure is quite unstable, particularly when RecA protein is removed (West et al 1981;Rao et al 1991. Attempts have been made to make the structure stable enough to withstand various subsequent manipulations (Fujiwara et al 1998a,b;Potaman et al 2002;Roulon et al 2002).…”