Stable three-stranded DNA made by RecA protein.

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 filament, 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 greater than proximal joints much greater than 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 resistant 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.     

Homologous recognition promoted by RecA protein via non-Watson-Crick bonds between identical DNA strands.

The RecA protein of Escherichia coli forms a nucleoprotein filament that promotes homologous recognition and subsequent strand exchange between a single strand and duplex DNA via a three-stranded intermediate. Recognition of homology within three-stranded nucleoprotein complexes, which is probably central to genetic recombination, is not well understood as compared with the mutual recognition of complementary single strands by Watson-Crick base pairing. Using oligonucleotides, we examined the determinants of homologous recognition within RecA nucleoprotein filaments. Filaments that contained a single strand of DNA recognized homology not only in a complementary oligonucleotide but also in an identical oligonucleotide, whether their respective sugar-phosphate backbones were antiparallel or parallel, and a filament that contained duplex DNA showed the same polymorphic versatility in the recognition of homology. Recognition of self by a filament that contains a single strand reveals that RecA filaments can recognize homology via non-Watson-Crick hydrogen bonds. Recognition of multiple forms of the same sequence by duplex DNA in the filament shows that it primarily senses base-sequence homology, and suggests that recognition can be accomplished prior to the establishment of new Watson-Crick base pairs in heteroduplex products. However, unlike the initial recognition of homology, strand exchange is stereospecific, requiring the proper antiparallel orientation of complementary strands.     

Resolution of an early RecA-recombination intermediate by a junction-specific endonuclease

The nucleoprotein filament formed on a circular single strand by Escherichia coli RecA protein in vitro can pair with homologous duplex DNA even when the latter lacks a free homologous end, but subsequent progression of the reaction through strand exchange requires an end in at least one strand of the duplex DNA. We purified from E. coli an endonuclease activity that cleaves the outgoing strand of duplex DNA at the junction of homologous and heterologous sequences in three-stranded RecA-recombination intermediates. This endonuclease activity also cleaves specifically at the junctions of duplex and single-stranded regions in synthetic double-stranded oligonucleotides whose central portion consists of unpaired heterologous sequences. These activities are consistent with a role in recombination and repair of DNA.     

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A small middle dotT base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a "synaptic pathway" involving a three-stranded nucleoprotein intermediate, rather than by a "helicase pathway" involving the separation and reannealing of DNA strands.     

Formation of base triplets by non-Watson-Crick bonds mediates homologous recognition in RecA recombination filaments.

Whereas complementary strands of DNA recognize one another by forming Watson-Crick base pairs, the way in which RecA protein enables a single strand to recognize homology in duplex DNA has remained unknown. Recent experiments, however, have shown that a single plus strand in the RecA filament can recognize an identical plus strand via bonds that, by definition, are non-Watson-Crick. In experiments reported here, base substitutions had the same qualitative and quantitative effects on the pairing of two identical strands in the RecA filament as on the recognition of duplex DNA by a third strand, indicating that similar non-Watson-Crick interactions govern both reactions.     

Ability of RecA protein to promote a search for rare sequences in duplex DNA.

RecA nucleoprotein filaments found homologous targets even when the latter was mixed with 200,000 times as much heterologous duplex DNA. By contrast, mixing of the single-stranded probe with only 100 times as much heterologous single strands markedly reduced the rate of finding homologous duplex molecules. Titration of the reaction with different proportions of homologous single-stranded DNA distinguished a condition under which the search for homology itself was rate limiting from a condition under which some later step was limiting. Less than 1 min was required to scan 6.4 kilobase pairs of duplex DNA for homology to a RecA-coated single strand of the same size, but these experiments revealed that rapid searching by RecA nucleoprotein filaments was largely confined to neighboring duplex molecules. These observations provide guidelines for the use of RecA protein in locating rare sequences in complex mixtures of duplex DNA, and we describe a simple protocol by which rare sequences can be rapidly enriched at least a thousandfold.     

The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA.

A key step in homologous recombination is the alignment and pairing of homologous DNAs. The Escherichia coli RecA protein initiates pairing by binding to single-strand DNA, forming a helical nucleoprotein filament. We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-[gamma-thio]triphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA. RecA can pair as few as eight bases of homology to form such synaptic complexes. The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs. Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search.     

The RecA proteins of Deinococcus radiodurans and Escherichia coli promote DNA strand exchange via inverse pathways

The RecA protein of Escherichia coli, and all filament-forming homologues identified to date, promote DNA strand exchange by a common, ordered pathway. A filament is first formed on single-stranded DNA, followed by uptake of the duplex substrate. These proteins are thereby targeted to single-strand gaps and tails where recombinational DNA repair is required. The observed course of DNA strand exchange promoted by the RecA protein from the extremely radioresistant bacterium Deinococcus radiodurans is the exact inverse of this established pathway. This reaction lies at the heart of a remarkably efficient system for the repair of DNA damage.     

RecA tests homology at both pairing and strand exchange

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. To observe the effects of mismatches on the kinetics of the RecA-catalyzed recombination reaction, we used assays based upon fluorescence energy transfer that can differentiate between the pairing and strand displacement phases. Oligonucleotide sequences that produced 2–14% mismatches in the heteroduplex product of strand exchange were tested, as well as completely homologous and heterologous sequences. The equilibrium constant for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and an increase in the rate of dissociation of the intermediates. In addition, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches. The equilibrium constant for pairing and the rate constant for strand displacement both decreased 6-fold as the heterology increased to 14%. These results suggest that discrimination of homology from heterology occurs during both pairing and strand exchange.     

Polarity of DNA strand exchange promoted by recombination proteins of the RecA family

Homologs of Escherichia coli RecA recombination protein, which have been found throughout the living kingdom, promote homologous pairing and strand exchange. The nucleoprotein filament, within which strand exchange occurs, has been conserved through evolution, but conservation of the polarity of exchange and the significance of that directionality has not been settled. Using oligonucleotides as substrates, and assays based on fluorescence resonance energy transfer (FRET), we distinguished the biased formation of homologous joints at either end of duplex DNA from the subsequent directionality of strand exchange. As with E. coli RecA protein, the homologous Rad51 proteins from both Homo sapiens (HsRad51) and Saccharomyces cerevisiae (ScRad51) propagated DNA strand exchange preferentially in the 5′ to 3′ direction. The data suggest that 5′ to 3′ polarity is a conserved intrinsic property of recombination filaments.     

The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.

The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound to the RecA protein primary site. We conclude that the specificity reflects an intrinsic property of the secondary site of RecA protein rather than an interaction between DNa molecules within nucleoprotein filament--i.e., self-recognition. The secondary DNA binding site displays a higher affinity for ssDNA than for double-stranded DNA, and the binding of ssDNA to the secondary site strongly inhibits DNA strand exchange. We suggest that the secondary binding site has a dual role in DNA strand exchange. During the homology search, it binds double-stranded DNA weakly; upon finding local homology, this site binds, with higher affinity, the ssDNA strand that is displaced during DNA strand exchange. These characteristics facilitate homologous pairing, promote stabilization of the newly formed heteroduplex DNA, and contribute to the directionality of DNA strand exchange.     

Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein.

To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.     

Polar branch migration promoted by recA protein: effect of mismatched base pairs.

Escherichia coli recA protein makes joint molecules from single-stranded circular phage DNA (viral or plus strand) and homologous linear duplex DNA by a polar reaction that displaces the 5' end of the plus strand from the duplex molecule [Kahn, R., Cunningham, R. P., DasGupta, C. & Radding, C. M. (1981) Proc. Natl. Acad. Sci. USA 78, 4786-4790]. Growth of the heteroduplex joint, which results from strand exchange or branch migration, stopped at the borders of regions of nonhomologous DNA that were variously located 145, 630, or 1202 nucleotides from the end. Accumulation of migrating branches at heterologous borders demonstrates that their migration is not the result of random diffusion but is actively driven by recA protein. Growth of the heteroduplex joint was blocked even when a heterologous insertion was located in the single-stranded DNA, a case in which the flexible single-stranded region might conceivably fold out of the way under some condition. The recA protein did not make joint molecules from phage phi X174 and G4DNAs, which are 70% homologous, but did join phage fd and M13DNAs, which are 97% homologous. In the latter case, heteroduplex joints extended through regions containing isolated mismatched base pairs but stopped in a region where the fd and M13 sequences differ by an average of 1 base pair in 10. These results suggest that in genetic recombination the discrimination of perfect or near-perfect homology from a high degree of relatedness may be attributable in part to the mechanism by which recA protein promotes strand transfer.     

Polarity of heteroduplex formation promoted by Escherichia coli recA protein.

When recA protein pairs circular single strands with linear duplex DNA, the circular strand displaces its homolog from only one end of the duplex molecule and rapidly creates heteroduplex joints that are thousands of base pairs long [DasGupta, C., Shibata, T., Cunningham, R. P. & Radding, C. M. (1980) Cell 22, 437-446]. To examine this apparently polar reaction, we prepared chimeric duplex fragments of DNA that had M13 nucleotide sequences at one end and G4 sequences at the other. Circular single strands homologous to M13 DNA paired with a chimeric fragment when M13 sequences were located at the 3' end of the complementary strand but did not pair when the M13 sequences were located at the 5' end. Likewise circular single-stranded G4 DNA paired with chimeric fragments only when G4 sequences were located at the 3' end of the complementary strand. To confirm these observations, we prepared fd DNA labeled only at the 5' or 3' end of the plus strand, and we examined the susceptibility of these labeled ends to digestion by exonucleases when joint molecules were formed. Eighty percent of the 5' label in joint molecules became sensitive to exonuclease VII. Displacement of that 5' end by recA protein was concerted because it did not occur in the absence of single-stranded DNA or in the presence of heterologous single strands. By contrast, only a small fraction of the 3' label became sensitive to exonuclease VII or exonuclease I. These observations show that recA protein forms heteroduplex joints in a concerted and polarized way.     

Isolation and visualization of active presynaptic filaments of recA protein and single-stranded DNA.

In the presence of ATP, recA protein forms a presynaptic complex with single-stranded DNA that is an obligatory intermediate in homologous pairing. Presynaptic complexes of recA protein and circular single strands that are active in forming joint molecules can be isolated by gel filtration. These isolated active complexes are nucleoprotein filaments with the following characteristics: (i) a contour length that is at least 1.5 times that of the corresponding duplex DNA molecule, (ii) an ordered structure visualized by negative staining as a striated filament with a repeat distance of 9.0 nm and a width of 9.3 nm, (iii) approximately 8 molecules of recA protein and 20 nucleotide residues per striation. The widened spacing between bases in the nucleoprotein filament means that the initial matching of complementary sequences must involve intertwining of the filament and duplex DNA, unwinding of the latter, or some combination of both to equalize the spacing between nascent base pairs. These experiments support the concept that recA protein first forms a filament with single-stranded DNA, which in turn binds to duplex DNA to mediate both homologous pairing and subsequent strand exchange.     

Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins.

The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA. In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay. Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP. These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.     

Direct observation of twisting steps during Rad51 polymerization on DNA

The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5°. Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 Å per base pair) and untwisted by 18.2° per base pair. They also confirm a stoichiometry of 3–4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65° ± 5°. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.     

3'' homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli.

The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro. The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell. Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle. Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA.     

Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis

SOS mutagenesis in Escherichia coli requires DNA polymerase V (pol V) and RecA protein to copy damaged DNA templates. Here we show that two distinct biochemical modes for RecA protein are necessary for pol V-catalyzed translesion synthesis. One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass. A separate RecA mode is necessary for translesion synthesis. The RecA1730 mutant protein, which was identified on the basis of its inability to promote pol V (UmuD'(2)C)-dependent UV-mutagenesis, appears proficient for the first mode of RecA action but is deficient in the second mode. Data are presented suggesting that the two RecA modes are "nonfilamentous". That is, contrary to current models for SOS mutagenesis, formation of a RecA nucleoprotein filament may not be required for copying damaged DNA templates. Instead, SOS mutagenesis occurs when pol V interacts with two RecA molecules, first at a 3' primer end, upstream of a template lesion, where RecA mode 1 stimulates pol V activity, and subsequently at a site immediately downstream of the lesion, where RecA mode 2 cocatalyzes lesion bypass. We posit that in vivo assembly of a RecA nucleoprotein filament may be required principally to target pol V to a site of DNA damage and to stabilize the pol V-RecA interaction at the lesion. However, it is only a RecA molecule located at the 3' filament tip, proximal to a damaged template base, that is directly responsible for translesion synthesis.     

RecA.oligonucleotide filaments bind in the minor groove of double-stranded DNA.

Escherichia coli RecA protein, in the presence of ATP or its analog adenosine 5'-[gamma-thio]triphosphate, polymerizes on single-stranded DNA to form nucleoprotein filaments that can then bind to homologous sequences on duplex DNA. The three-stranded joint molecule formed as a result of this binding event is a key intermediate in general recombination. We have used affinity cleavage to examine this three-stranded joint by incorporating a single thymidine-EDTA.Fe (T*) into the oligonucleotide part of the filament. Our analysis of the cleavage patterns from the joint molecule reveals that the nucleoprotein filament binds in the minor groove of an extended Watson-Crick duplex.