(Received for publication, October 21, 1994)
From the
A RecA protein-generated triple-stranded DNA species can be observed by electron microscopy, within narrowly defined conditions. Three-stranded DNA is detected only when initiation of normal DNA strand exchange is precluded by heterologous sequences within the duplex DNA substrate, when ATP is hydrolyzed, and when the DNA is cross-linked with a psoralen derivative prior to removal of RecA filaments. When adenosine 5`-O-(thiotriphosphate) is used, only the product hybrid duplex DNA can be cross-linked within the RecA filament. The third strand is either displaced or interwound in a conformation that does not permit cross-linking. When ATP is hydrolyzed by RecA, all three strands are cross-linked within the filament in a complex pattern that suggests a dynamic structure. This structure is altered when RecA protein is removed before cross-linking. Hsieh et al.(1990) and Rao et al.(1991, 1993) have proposed, on the basis of nuclease protection and chemical modification studies, that a stable triple-stranded DNA species can persist after removal of RecA protein. We have been unable to visualize these triple-stranded structures by the methods used in the present investigation. When RecA removal was followed immediately by interstrand cross-linking, only the two strands of the hybrid duplex DNA were cross-linked.
RecA protein is essential for recombinational DNA repair and
homologous recombination in Escherichia coli. It promotes a
DNA strand exchange reaction in vitro involving circular
single-stranded DNA (ssDNA) ()and homologous linear duplex
DNA. Studies (Roca and Cox, 1990; Kowalczykowski, 1991; Radding, 1991;
Cox, 1994) (
)have provided evidence for up to four reaction
phases. The first phase is the cooperative assembly of RecA on ssDNA.
In the presence of ATP or ATP
S (a nonhydrolyzable ATP analog),
this results in a right-handed helical filament with a pitch of 95
Å and an axial rise per nucleotide of 5.1 Å. There are 6
RecA monomers and 18 nucleotides per helical turn. The second phase is
the homologous alignment of a duplex DNA with the ssDNA in the
nucleoprotein filament. When the topology of the DNA substrates
permits, the second phase is followed closely by the transfer of the
complementary strand of the duplex DNA to the ssDNA to form a short
(
1000 bp) region of hybrid or heteroduplex DNA. (
)This
third phase is facilitated by binding energy within the filament and
requires bound ATP but not ATP hydrolysis (Menetski, et al.,
1990; Rehrauer and Kowalczykowski, 1993). The limited strand exchange
in phase 3 is bidirectional, blocked by structural barriers in the DNA,
and limited to three DNA strands. In the final phase, this short
segment of hybrid DNA is extended in a branch migration coupled to ATP
hydrolysis (Cox, 1994). The extension is unidirectional (5` to 3` with
respect to the single-stranded DNA) (Konforti and Davis, 1992; Jain et al., 1994), bypasses structural barriers in the DNA (Kim, et al., 1992a; Rosselli and Stasiak, 1991), and accommodates
four DNA strands (Kim, et al., 1992b).
The mechanism by which RecA facilitates the alignment of homologous sequences in phase 2 is being addressed in several laboratories. RecA binds to the phosphate backbone of the ssDNA, leaving the bases exposed and accessible for base pairing in the helical groove of the RecA filament (Di Capua and Müller, 1987; Dombroski et al., 1983; Leahy and Radding, 1986). Structural studies and nucleolytic protection experiments have demonstrated that there are distinct DNA binding sites within the RecA filament that can simultaneously bind up to three DNA strands (Chow et al., 1988; Kubista et al., 1990; Pugh and Cox, 1987a, 1987b; Takahashi et al., 1989, 1991).
Two types of models have been postulated for the mechanism by which RecA mediates homologous recognition between two DNA molecules. These can be loosely summarized as ``pairing before strand separation'' versus ``strand separation before pairing'' (Stasiak, 1992). A triplex DNA species could be an intermediate in either type of model (Fig. 1).
Figure 1: Alternative models for DNA pairing during RecA-mediated DNA strand exchange. Pathway1, a pathway involving formation of a ``pre-strand switch'' triplex DNA; pathway2, pathways involving a concurrent alignment and strand switch. A ``post-strand switch'' triplex DNA is a plausible intermediate in both cases.
Type I models arise from ideas originally set forth by Howard-Flanders and colleagues(1984) and propose that a novel DNA triplex structure exists as the initial pairing intermediate in RecA protein-promoted DNA strand exchange reactions (Howard-Flanders et al., 1984; Hsieh, et al., 1990). In the scheme above, this triplex would be the product of phase 2. The triplex DNA would be a ``pre-strand switch'' triplex, with the two strands of the substrate duplex still joined by Watson-Crick base pairs and the ssDNA in the RecA filament interacting via non-Watson-Crick hydrogen bonds. The strand switch could result in formation of a distinct ``post-strand switch'' triplex, in which the strands of the hybrid DNA would be Watson-Crick base paired and the nascent-displaced strand would interact via non Watson-Crick pairing.
In type II models, homologous recognition and DNA strand exchange (phases 2 and 3 above) are concurrent (Adzuma, 1992). A local strand separation in the duplex DNA allows base pairing between the complementary strand of the duplex DNA and the incoming single-stranded DNA within the RecA filament. The binding energy within the filament effectively stabilizes the product hybrid DNA. Alternative plausible fates for the third strand lead to variations in type II models. The third strand could be immediately displaced from the RecA filament, it could remain transiently wound around the product duplex but not interact with it, or it could be transiently hydrogen-bonded to the hybrid duplex in the major groove. The latter case gives rise to the possibility that a triplex DNA could be a discrete intermediate in a type II pathway. Any type II triplex, however, would be a ``post-strand switch'' triplex.
Regardless of when it is formed, a RecA-mediated triplex DNA structure must have parallel like strands and be sequence independent as long as the DNAs are homologous. These factors distinguish it from the well characterized stable DNA triplexes that are formed non-enzymatically (Hanvey et al., 1988; Moser and Dervan, 1987). The RecA-generated species has been called recombinant (or R-form) DNA. A triple-stranded DNA that may be analogous to R-form DNA but is formed in the absence of proteins has recently been reported (Shchyolkina et al., 1994).
Indirect measurements have revealed that thousands of base pairs of homology can be aligned at early stages of the DNA strand exchange reaction (Bianchi et al., 1983; Schutte and Cox, 1987, 1988). Electron microscopy with psoralen-photocross-linked RecA synaptic complexes has provided unambiguous evidence for the alignment and close juxtaposition of three DNA strands over thousands of base pairs (Bortner and Griffith, 1990; Umlauf et al., 1990). However, these approaches do not distinguish between type I or type II models to the extent that three strands could be transiently juxtaposed within the filament in either case.
Chemical modification of synaptic complexes formed in the
presence of ATPS confirmed that the base pairing within the
parental dsDNA is disrupted. One strand of the duplex DNA is
Watson-Crick base paired to the ssDNA within the RecA filament, and the
homologous strand is displaced (Adzuma, 1992). This work favors a type
II model, at least when ATP
S is present. Characterization of
deproteinized DNA pairing intermediates by nucleolytic digestion,
chemical modification, and thermal stability studies has provided
evidence for the presence of a stable triplex DNA that has many of the
characteristics of a post-strand switch triplex (Rao et al.,
1991, 1993; Chiu et al. 1993; Burnett et al., 1994).
No evidence that uniquely favors the existence of a pre-strand switch
triplex has been reported.
There is no shortage of models depicting
what an R-form triplex might look like. Recent evidence involving base
analog-substituted substrate duplex DNAs in RecA-mediated DNA strand
exchange reactions and chemical modification of putative three-stranded
DNA structures in the presence or absence of RecA filaments reveals
that hydrogen bonding to the N-7 of guanine and adenine is not crucial
in stabilization of any intermediate in DNA strand exchange (Adzuma,
1992; Jain et al., 1992; Rao et al., 1993; Jain,
1994). In contrast, Hoogsteen base pairing to the N-7 of purines is
essential for non-enzymatic triplex DNA formation (Hanvey et
al., 1988; Moser and Dervan, 1987). ()Umlauf(1990)
proposed that triple-stranded recombination intermediates utilize two
additional hydrogen bonds between the Watson-Crick base pairs in the
major groove of a duplex DNA and a paired third strand (Umlauf, 1990).
The effects of enzymatic methylation of the N-6 of adenine and N-4 of
cytosine on the stability of deproteinized triple-stranded
intermediates support the involvement of Watson-Crick positions in base
triplets (Rao et al., 1993). Models similar to the Umlauf
proposal have recently been refined on theoretical grounds (Zhurkin et al., 1994).
The present studies extend the work of Umlauf et al.(1990). They examine the effects of ATP hydrolysis on formation of three-stranded DNA that can be cross-linked with psoralen derivatives and explore the nature of the structure present after deproteinization of the complexes. They were carried out to provide direct visual support for the triple-stranded DNAs characterized in many previous studies using electron microscopy. While a three-stranded DNA species can be visualized after the DNA is cross-linked within the RecA filament, we were unable to confirm the existence of a stable triplex DNA after RecA removal. Cross-linking of three strands can only occur within the filament under narrowly defined conditions.
Reactions involving
ATPS were performed at 37 °C in a standard reaction buffer
containing 25 mM Tris acetate (80% cation, pH 7.5), 4 mM magnesium acetate, and 1 mM dithiothreitol.
Single-stranded DNA (10 µM) was preincubated with SSB (0.9
µM) for 10 min before addition of RecA protein (6.0
µM) and ATP
S (1 mM). After incubation of
this mixture for 10 min, the reaction was initiated with duplex DNA
(11.4 µM). Conditions for ATP and ATP
S reactions are
different so that each proceeds optimally. The ssDNA molecules are in
2-fold molar excess with respect to the dsDNA molecules in all
reactions.
Some samples were deproteinized
prior to cross-linking. Aliquots (20 µl) of the reaction mixtures
(60-min time points) were incubated with Proteinase K (1 mg
ml) and SDS (0.05%), Triton X-100 (0.5%), Nonidet
P-40 (1%), N-laurolylsarcosine (0.1%), or deoxycholic acid
(0.1%) at 37 °C for 15 min. All concentrations given are final
concentrations after addition of all components. In some cases,
detergent was not added, and deproteinization with proteinase K (1 mg
ml
) was carried out 5 s, 1 min, or 2 min prior to
addition of AMT as indicated in the specific experiment. The samples
were immediately photocross-linked with AMT, dialyzed, and spread for
electron microscopy as described above.
The cross-linking density resulting from psoralen-long-wave UV irradiation was determined using RecA-coated gapped circles, which contained a 2,159-kbp double-stranded region. After the psoralen treatment, these molecules were deproteinized as described above and then completely denatured by heating at 70 °C for 15 min in 20 mM NaCl, 5 mM EDTA, 5% HCHO. Samples were then cooled on ice prior to spreading. When cross-linked DNA is completely denatured, each cross-over of the single strands can be assumed to represent a cross-link. Examination of such molecules by electron microscopy therefore allows an approximate determination of the cross-linking density between duplex strands. In the present case, the cross-linking densities were high enough to often produce cross-links close enough together that the denatured material appeared to contain undenatured segments (the limit of sample resolution is such that ssDNA and dsDNA cannot be differentiated if segments are less than 50 bp in length. To allow for this effect in the determination of the cross-linking density, the size of ``undenatured'' regions were determined by measurement and a correction applied as previously described (Schnös and Inman, 1987). Our estimate for the cross-linking density resulting from AMT treatment of dsDNA within a RecA filament is about 10 cross-links/kbp. This is reduced to 3.8 cross-links/kbp in dsDNA treated with TMP in absence of RecA.
Figure 2: DNA substrates used in this study. The numbers denote the size of the M13 mp8 single-strand circle or the length of the homologous (thinlines) and heterologous (thicklines) regions in the duplex DNA substrates. At the heterologous end of each duplex molecule, there are very short sequences with homology to M13 mp8 (not shown), which total 7 (a) and 19 (b) bp. Neither of these regions is long enough to take part in DNA pairing reactions under the conditions used for these experiments.
Figure 3: The homologous strand of the duplex DNA is displaced in paired DNA intermediates. RecA-mediated DNA strand exchange reactions were carried out in the presence of ATP with circular ssDNA (10 µM), linear dsDNA with 5` or 3` homology (11.4 µM), 6.6 µM RecA, and 2 µM SSB as described under ``Materials and Methods.'' The samples were prepared and visualized by electron microscopy. A, a representative paired complex involving linear dsDNA with proximal homology; B, a representative paired intermediate involving linear dsDNA with distal homology. The double-stranded circular DNA corresponds to the hybrid DNA formed in the strand exchange reaction, and the long single strand represents the displaced homologous strand. The duplex tail reflects the unreacted DNA within the duplex substrate.
With homology at the proximal end of the duplex DNA,
the cross-linked joint molecules appeared similar to the uncross-linked
intermediates. There was no indication of any triple-stranded DNA
structures in any of the 62 paired complexes observed in two
independent experiments (Fig. 4A). The joint molecules
represented 95% of the total molecules (duplex DNA substrates and
paired complexes) examined. A similar result, the absence of
triple-stranded DNA in joint molecules with hybrid DNA extending to the
proximal end, was obtained after only 15 min of reaction (data not
shown). ()
Figure 4: Electron micrographs of AMT cross-linked DNA intermediates. DNA strand exchange reactions were carried out in the presence of ATP as described under ``Materials and Methods.'' A, cross-linked DNA intermediates formed with circular single-stranded DNA and duplex DNA with proximal homology; B-E, cross-linked paired DNA intermediates involving circular ssDNA and duplex DNA with distal homology. Three representative paired complexes formed in DNA strand exchange reactions that proceed from the 3` homologous free end of the duplex DNA are shown in B-D. Although the DNA strand paths in these molecules are complex, there are many consecutive regions that are consistent with a triple-stranded structure. The inset in B represents our interpretation of one of these triple-stranded regions between the twoarrows, with multiple cross-links between the single-stranded DNA and the duplex DNA. Pairing of the ssDNA with an internal homologous region in the duplex DNA is depicted in E.
In contrast, when homology was at the distal end of the duplex DNA, the AMT-cross-linked joint molecules consisted of three types. The first class of intermediates (25% of the paired complexes observed) were indistinguishable from the uncross-linked intermediates shown in Fig. 3B (Table 1). These intermediates were observed in 12 independent experiments; the detailed counts in Table 1come from three of these, one chosen at random from experiments carried out with each of three different psoralen derivatives.
The second type of paired complexes contained regions of triple-stranded DNA extending to the end of the duplex DNA substrate (Fig. 4, panelsB-D). The structures are complicated, and it is difficult to unambiguously trace the path of the three strands in most of the triple-stranded DNAs. The three-stranded DNA contained alternating substructures in which all three-DNA strands appear to be aligned, in which one strand is displaced from a duplex DNA region and in which all strands are single stranded. The inset within Fig. 4B shows our interpretation of a region in the micrograph showing these features. The spreading conditions used to prepare the molecules shown in Fig. 4do not cause significant denaturation of normal dsDNA. However, in the triple-stranded regions, there were repeated examples of short regions where all three strands appeared to be in a single-stranded form. This suggests that the triple-stranded structure was in a strained conformation within the filament and unraveled to the extent permitted by the cross-linking upon RecA removal. These triple-stranded intermediates represented 56% of the paired DNA molecules (Table 1).
The third class of synaptic complexes were three-stranded structures identical to the second, except that they involved pairing of the circular ssDNA with an internal region of homology within the duplex DNA (Fig. 4E). They were similar to the three-stranded structures previously characterized by Umlauf et al.(1990). These intermediates represented 19% of the paired complexes observed (Table 1).
The stabilization of
the triple-stranded structures formed in RecA-mediated DNA strand
exchange reactions is dependent on AMT cross-linking. As we decreased
the psoralen concentration 20-fold (1.5 µg ml final concentration), the fraction of intermediates in classes 2
and 3 decreased significantly. Photocross-linking with a 100-fold lower
concentration of AMT (0.3 µg ml
final
concentration) almost eliminated detection of the three-stranded DNA
structures (data not shown).
Very similar results were obtained with two other psoralen derivatives, TMP and HMT (Table 1). These have lower solubilities and weaker affinities for duplex DNA than AMT (Isaacs et al., 1977).
For the
ATPS-mediated reactions, cross-linking with AMT prior to
deproteinization had no effect on the results. All of the DNA
intermediates contained displaced homologous single strands, whereas
the hybrid duplex DNA regions were cross-linked. There was no
indication of triple-stranded intermediates in any of the 95 and 75
paired complexes involving duplex DNA with proximal or distal homology,
respectively, in two independent experiments for each reaction (data
not shown). The total number of molecules counted in the experiments
described above were 125 and 108, respectively. No triple-stranded DNA
was observed in any reaction carried out with ATP
S, with or
without DNA cross-linking.
We disrupted the RecA filament on the paired intermediates by treatment with Proteinase K and either of two nonionic detergents, Triton X-100 or Nonidet P-40, or one of two anionic detergents, N-laurolylsarcosine or deoxycholic acid, for 15 min at 37 °C. Samples were then immediately cross-linked with AMT. All of the paired intermediates contained displaced single DNA strands, and no triple-stranded DNA was observed. The results with the four detergents were similar and were combined in Table 2. To verify the presence of cross-linking within the duplex DNA regions of paired complexes and to confirm that the cross-linking reactions worked under the conditions used, individual reaction mixtures were heat denatured after deproteinization and AMT cross-linking. DNAs that were incubated with Proteinase K and any of the four detergents revealed degrees of cross-linking in the duplex DNA similar to those observed without the detergents. Control experiments had shown that SDS interfered with the cross-linking reaction (data not shown).
We wanted to examine the transition of triple-stranded DNA recombination intermediates detected within the filaments to complexes with displaced single-stranded DNA tails. Reaction mixtures involving circular ssDNA and linear dsDNA with distal homology were treated with Proteinase K for 5 s (psoralen added immediately after addition of Proteinase K), 1 min, or 2 min and then cross-linked with AMT. Triple-stranded structures were observed in half of the paired complexes counted when the DNAs were cross-linked at 5 s (Table 2). Incubation with Proteinase K for 1 min significantly reduced the number of three-stranded complexes, and 2 min of digestion almost eliminated the triple-stranded structures (Table 2). Evidently, removal of RecA leads to an altered triple-stranded structure that is not cross-linkable, or the triple-stranded structure is lost immediately because the third strand is no longer found to be associated with the hybrid DNA. The DNA intermediates appeared to be dotted with remnants of the RecA filament as observed by electron microscopy (data not shown). We verified that the duplex DNA regions within the paired complexes were cross-linked in the presence of Proteinase K, so the results do not reflect an inhibition of the cross-linking process.
As previously reported (Umlauf et al., 1990), a triple-stranded DNA species, reflecting homologous pairing between a single strand and a duplex DNA, can be cross-linked within a RecA filament and visualized by electron microscopy. The present study demonstrates that this species can be detected only in a limited set of circumstances. The three-stranded DNA is found only when complete DNA strand exchange is precluded (e.g. by heterologous sequences at the proximal end of the linear duplex DNA). The triple-stranded DNA is also observed only when ATP is hydrolyzed and cannot be detected once the RecA filament is removed. No three-stranded DNA that can be cross-linked with AMT is observed by electron microscopy during normal DNA strand exchange reactions between completely homologous DNA substrates or when pairing is restricted to the proximal end of the duplex DNA substrate.
There is one major point of agreement among
all of the studies in which the status of individual DNA strands within
RecA-paired complexes could be assessed. Under most conditions, the
RecA filament tends to stabilize the products of DNA strand
exchange. When ATPS was employed in the present study, only the
hybrid duplex DNA was cross-linked within paired regions at either end
of the duplex DNA. This result is in close agreement with that of
Adzuma(1992). When pairing occurred on the proximal end of the duplex
DNA, the hybrid DNA was cross-linked exclusively even when ATP was
hydrolyzed. The homologous strand of the duplex is evidently displaced
rapidly during strand exchange with ATP if the structure of the DNA
substrates permits.
We saw no evidence for triple-stranded structures after deproteinization using the methods described in this investigation. Only the hybrid duplex can be cross-linked with AMT in the deproteinized intermediates. The characterization of these deproteinized species with chemical and enzymatic probes also indicates that the two strands representing the hybrid duplex are joined by Watson-Crick base pairs (Rao et al., 1991; Chiu et al., 1993; Rao et al., 1993; Burnett et al., 1994). The third strand in the stable triplexes may be hydrogen-bonded in the major groove of this product duplex DNA (Rao et al., 1993) but not in a conformation suitable for cross-linking with a psoralen derivative.
It is worth pointing out that there is no direct evidence in any study for a DNA triplex existing prior to the actual strand switch. In all of the putative triplex DNA structures described to date for RecA reactions, including the stable triplexes detected after deproteinization, the Watson-Crick pairing occurs between the strands destined to form the hybrid DNA product. In effect, there is no evidence for a type I model. Triplex DNA described to date appears to be a transient product of DNA strand exchange, existing only until the displaced strand can be unraveled from the product duplex and persisting only when that unraveling is precluded by DNA structure. The relationship of this species to the process by which two DNA molecules are initially aligned is unclear.
The
cross-linking patterns observed in the circumstances where we do
observe three-stranded DNA suggest a dynamic structure (Fig. 5).
The single minus strand in the complex may be positioned so that it can
pair alternatively with either of the complementary plus strands.
Psoralen and its derivatives most readily cross-link antiparallel
strands that are joined by Watson-Crick base pairs (Cimino et
al., 1985). The linking of three strands (when ATP is hydrolyzed
and the third strand cannot be displaced) suggests that the paired
region is divided into small subdomains in which the minus strand is
alternately paired with either complement. Such a pattern was
documented in Umlauf et al.(1990). The alternation could be
facilitated by ATP hydrolysis. When ATP is bound (or ATPS), the
hybrid duplex is favored. ATP hydrolysis may lead to rapid displacement
of the third strand (Burnett et al., 1994). If the extra
strand cannot be displaced, bound ADP, or the absence of nucleotide,
may permit the minus strand to switch back and pair with its original
complement in the substrate duplex. This would yield a mixture of the
``pre-switched'' and ``post-switched'' states, in
the terminology of Burnett et al.(1994).
Figure 5: A model for DNA pairing within a RecA filament that is hydrolyzing ATP. The single minus(-) strand alternates between complementary (+) strand partners without formation of a base-paired triplex DNA.
This kind of switching by the minus strand could be viewed as a sampling mechanism in the homology search required to align the two DNA molecules. However, DNA pairing occurs efficiently without ATP hydrolysis (at least in vitro). The switching may instead reflect conformation changes in a relatively tight complex that would normally lead to displacement of the third strand. The third strand is at least transiently displaced in distal joints because it is susceptible to exonuclease I digestion (Bedale et al., 1993). The same process may contribute to dissociation of DNA joints that are not productive (Burnett et al., 1994).
A triplex species in which the bound single-stranded DNA interacts with the major groove of the incoming duplex DNA still seems like a plausible, if not probable scenario for DNA pairing. The lack of direct evidence for a ``pre-switch'' triplex could simply reflect a short lifetime, with the strand switch to hybrid DNA occurring rapidly once alignment is achieved. The like-strand pairing recently detected by Rao and Radding(1993) suggests that the two sites in the filament where the plus strands bind are close enough for these strands to interact.
We cannot rule out the possibility that a triple-stranded DNA structure remains after RecA removal. However, our inability to visualize triplex DNA after deproteinization does establish some additional structural parameters. First, the triple-stranded structure must be less stable under some or all conditions than duplex DNA. No uncross-linked triplex DNA species survives our spreading procedures, even though the same procedures do not disrupt duplex DNA. Although the triplex DNA has been reported to be thermally stable (Hsieh et al., 1990; Rao et al., 1991, 1993), the surface tension to which DNA is subjected during spreading might preferentially destabilize uncross-linked triplex DNA. We note that the R-form DNA described by Shchyolkina et al.(1994) is much less stable than normal duplex DNA. Second, the cross-linking of the hybrid duplex DNA strands after deproteinization indicate that these strands are Watson-Crick base paired, consistent with chemical modification patterns recently reported (Chiu et al., 1993). The third strand is clearly not in a conformation that permits it to be AMT-cross-linked to either of the other two strands. Finally, the change in cross-linking patterns brought about by deproteinization also allows us to conclude that the structure of the DNA species present after deproteinization is not the same as the three-stranded DNA pairing intermediate that can be cross-linked within the RecA filament. Whatever its detailed structure, the relationship of the deproteinized species to events that occur within the RecA filament remains to be determined.