(Received for publication, June 10, 1994; and in revised form, December 16, 1994)
From the
We have examined coupled reactions with the RecA protein of Escherichia coli, which can mediate DNA strand exchange in vitro between homologous DNA molecules, and the RecJ exonuclease, a 5` to 3` single-stranded DNA exonuclease. In RecA-mediated strand-transfer reactions between circular single-stranded and duplex linear DNA, we have found that RecJ stimulates the rate of heteroduplex product formation. Because RecJ must be present concurrent with strand transfer and RecJ does not detectably stimulate the synapsis stage of the reaction, we believe that RecJ stimulates specifically the branch migration phase of the RecA strand-transfer reaction. RecJ also dramatically enhances the efficiency with which RecA is able to traverse regions of non-homology in the substrates. We propose a model where RecJ degrades the displaced strand produced by strand exchange which competes for pairing with the transferred strand, thus driving forward the unidirectional branch migration mediated by RecA protein. This suggests a new role for exonucleases in genetic recombination, facilitating the strand-transfer reaction itself.
The exchange of homologous strands of DNA is a key step in
genetic recombination and repair. The process of in vitro strand transfer between homologous single-stranded and
double-stranded DNA promoted by the RecA protein of E. coli has been well characterized(1, 2, 3) .
The reaction is divided into three phases: (i) pre-synapsis, the
formation of a polar helical filament by RecA on single-stranded DNA
(ssDNA); ()(ii) synapsis, the rapid pairing of a
complementary strand of double-stranded DNA (dsDNA) with the
RecA-coated ssDNA; and (iii) strand exchange, the unidirectional branch
migration resulting in the formation of a new heteroduplex molecule and
a displaced single strand of DNA. Although RecA is the protein that
performs the actual mechanics of strand exchange, genetic recombination in vivo requires the participation of additional recombination
proteins(4) .
The recJ gene was identified as an essential gene for recombination and recombinational DNA repair pathways which operate independently of the recBCD genes(5, 6) . The recJ gene encodes a 5`- to 3`-exonuclease (7) highly specific for ssDNA. Exonucleases have been thought to play two roles in the process of genetic recombination. First, because RecA requires that one of the partners contains ssDNA at the region of homology in the strand-transfer reaction(8) , exonucleases may function to convert dsDNA to ssDNA to reveal this homology. RecJ could accomplish this function by acting in concert with a DNA helicase. Second, after strand transfer is complete, exonucleases may ``trim'' regions of DNA that remain unpaired to allow resealing of DNA strands. However, the ssDNA specificity of the RecJ exonuclease led us to consider a third role: the enhancement of the strand-exchange process itself. That is, the exonucleolytic degradation of a 5`-ssDNA displaced during the RecA-mediated strand exchange process might enhance the efficiency of homologous strand uptake by removing a competitor strand for pairing.
M13mp18 bacteriophage virion and replicative form (RF) DNA were purified from infected JM101 cells as described previously (7) with additional chromatography on HTP hydroxylapatite (Bio-Rad). The RF DNA was linearized by digestion with PacI (New England Biolabs) for use as a substrate in the RecA strand-transfer reactions. Experiments with substrates carrying nonhomologous regions employed M13mp18 with an insert of 187 bp (the BamHI SphI fragment of the tetA gene of plasmid pBR322). RF DNA from this derivative, ``M13-STL1,'' was cut with BamHI, SphI, or PacI, creating a linear dsDNA substrate which contains the 187-bp nonhomologous region at the 3` complementary end, the 5` complementary end, or 2.1 kb from the 3` complementary end, respectively, of the linear substrate.
For experiments examining the
effect of RecJ exonuclease on linear dsDNA, this substrate was
pretreated with RecJ exonuclease under the strand-exchange standard
reaction conditions by incubating linear dsDNA substrate for 1 h at 37
°C, omitting the ssDNA substrate. The DNA was deproteinized with
Proteinase K, phenol-extracted, and purified by gel filtration with
Bio-Gel P-60 (Bio-Rad). The DNA fractions were concentrated with a
Microcon-30 (Amicon) microcentrifuge filter and resuspended to a final
concentration of 100 µg/ml in ddHO.
For radiolabeled
reactions, linear dsDNA substrate was 3`-labeled with
[S]dATP (DuPont NEN) and terminal
deoxynucleotidyltransferase (Promega). The labeled DNA was
phenol-extracted, columnpurified, and concentrated as described above.
Substrates and products were separated on an agarose gel as described
above, after which the gel was placed on gel blot paper (Schleicher
& Schuell), dried for 1.5 h at 70 °C, and exposed to XAR5 film
(Kodak).
Because RecJ exonuclease requires a free ssDNA end,
neither of the starting substrate molecules in the strand-transfer
reaction are subject to RecJ digestion. However, as the strand-transfer
reaction progresses (Fig. 1), the displaced 5`
(+)-single-strand can be, in theory, subject to RecJ hydrolysis.
During the strand-transfer reaction, the 3`(-)-transferred strand
from the duplex has two potential pairing partners: the 5`
(+)-duplex strand and the (+)-single-strand circle.
Therefore, the digestion of the 5` (+)-partner by RecJ could
accelerate the uptake of the 3`(-)-strand on the single-strand
circle. It should be noted that RecA and SSB affect the activity of
RecJ protein. RecA bound to ssDNA inhibits RecJ while SSB has the
ability to enhance the exonuclease activity of the protein. ()
Figure 1: Model for RecA + RecJ-coupled action. RecA initiates strand exchange producing a displaced (+)-strand with a 5` end, a substrate for RecJ exonuclease. The removal of this competing strand may drive unidirectional branch migration and stimulate the rate of heteroduplex product formation.
We performed standard RecA strand-transfer reactions
between linear duplex DNA and circular ssDNA in the presence of SSB
protein, with and without the addition of RecJ exonuclease. Products
were examined by electrophoresis. In Fig. 2A, the left panel shows the progression of the RecA strand-exchange
reaction over a 90-min time course. Under these reaction conditions,
fully heteroduplex circular product formation was not seen until 60
min. In contrast, in the right panel, the time course for the
RecA + RecJ-coupled reactions appeared to show product formation
as early as 15 min. In the RecA + RecJ reactions, intermediates
appeared faster-migrating, consistent with the removal of the 5` tail
of a branched intermediate during the course of strand transfer (see Fig. 1). The time required for the RecA strand exchange to go to
completion is a result of using slightly suboptimal RecA concentrations
which enhance our ability to see the stimulation of the reaction by
RecJ. With optimal RecA conditions, RecA reactions show product
formation at 15 min. ()Under these conditions, stimulation
of the reaction by RecJ is still evident.
Figure 2:
RecJ stimulation of strand exchange. Time
points of strand-exchange reactions between M13mp18 circular ssDNA and
linear dsDNA, deproteinized with Proteinase K, and resolved on a 1%
agarose gel. A, reaction at left contains RecA and
that at right contains RecA + RecJ. Both reactions
included SSB protein. Product formation is determined by the appearance
of a band corresponding to nicked circular dsDNA standards run with
each gel. B, time points of strand-exchange reactions as in A, except that the linear dsDNA is 3`-labeled with
[S]dATP. Electrophoresis was performed as in A with the addition of 50 µg/ml ethidium bromide in the
gel and running buffer.
These effects are seen
more clearly in a similar set of reactions in which the linear duplex
substrate was 3`-end-labeled with [-
S]dATP (Fig. 2B). The autoradiogram of these reactions
highlights the increased rate of product formation as well as the
faster migrating branched intermediates (at 15 and 30 min) in the RecA
+ RecJ reaction (right panel). The linear single-strand
product is clearly visible in the RecA reaction (Fig. 2B, left panel) but is absent in the
coupled reaction. This would suggest that under these conditions RecJ
is capable of degrading this displaced strand. Also apparent at early
points in both the RecA and RecA + RecJ reactions (at 5 to 30 min)
is a relatively fast-migrating intermediate species (migrating faster
than circular duplex product). This early intermediate is a joint
molecule which can be converted to both substrates and products after
deproteinization and heat treatment,
but its exact
structure is not known.The short-lived nature of this intermediate and
some variability in the speed of the strand-transfer reactions may
account for its spurious presence.
The addition of RecJ did not
obviate the necessity for RecA or ATP in the strand-transfer reactions
under conditions described above. Only very small amounts
of RecJ protein need to be provided to observe stimulation: the
equivalent of 3-4 RecJ molecules per displaced 5` end of ssDNA.
In addition, although the above reactions were performed in the
presence of SSB, we have also observed RecJ stimulation of strand
exchange in the absence of SSB.
The efficiency of strand
exchange by RecA without SSB under these in vitro conditions
was severely reduced, presumably because of imperfect RecA filament
formation through regions of secondary
structure(14, 15) .
Figure 3: RecJ pretreatment of linear dsDNA substrate. Standard RecA reactions were performed between circular M13mp18 ssDNA and either linear RF dsDNA substrate (left panel) or linear dsDNA substrate pretreated with RecJ (right panel). Product formation is indicated by the appearance of a band corresponding to a nicked circular duplex.
Figure 4:
Joint molecule formation. Filter binding
assays for joint molecule formation were performed after RecA reactions
between M13mp18 circular ssDNA and S-labeled linear RF
dsDNA. Reactions contained RecA alone (
) and RecA + RecJ
(
).
With a 3` end 187-bp non-homology, no strand exchange is seen in the RecA reaction, and weak product formation is detected in the RecA + RecJ reaction (data not shown). Densitometric analysis of ethidium bromide-stained gels of deproteinized reactions allows us to estimate that approximately 15% of the linear molecules were converted to relaxed duplex circular products in the RecA + RecJ reactions; no detectable product was formed in reactions with RecA alone. Under these reaction conditions, RecA is unable to initiate strand exchange without a homologous 3` complementary end and the presence of RecJ in the reaction alleviates this inhibition to transfer only weakly.
In contrast, strand exchange between circular ssDNA and a linear substrate containing internal non-homology was more substantially enhanced by RecJ. The linear substrate in this experiment harbored 187 bp of non-homology 2.1 kb from the 3` complementary end of the dsDNA. In these reactions, strand exchange is assumed to proceed to the region of non-homology but further branch migration is apparently inhibited (19) . Reactions were monitored over a time course of 60 min. (Fig. 5) The reactions containing RecJ show significant product formation by 15 min, whereas there is only a faint corresponding band in the RecA reactions at this and later time points. Product formation is determined by the appearance of a band co-migrating with a marker formed by denaturing and reannealing the linear dsDNA and circular ssDNA substrates. The RecA reactions exhibit more slowly migrating species which we presume is a branched intermediate stalled at the point of non-homology. These results suggest that the action of the RecJ exonuclease stimulates the efficiency with which RecA traverses the region of non-homology. At later time points, increased amounts of slow-migrating ``aggregate'' DNA appears in the wells. These aggregates may be the result of secondary transfer events initiated from extruded ssDNA following product formation.
Figure 5: Stimulation of strand exchange through regions of non-homology. Time points of strand-exchange reactions between M13mp18 circular ssDNA and linear dsDNA with a 187-bp non-homologous region 2.1 kb from the 3` end of the complementary strand. Reactions were deproteinized with Proteinase K and resolved on a 0.8% agarose gel. Reaction at left contains RecA and that at right contains RecA + RecJ. Both reactions included SSB protein. Product formation was determined by the appearance of a band co-migrating with a marker formed by annealing the linear dsDNA substrate (with the nonhomologous region) to the M13mp18 circular ssDNA substrate.
As a further test to prove these product bands were truly the result of complete transfer, an independent set of reactions was monitored by restriction digestion of deproteinized samples with BglII and AlwNI. The linear substrate is cleaved by these two enzymes into 1.9-kb, 2.5-kb, and 3.0-kb fragments. As the transfer goes to completion, the 1.9-kb and 3.0-kb terminal fragments will disappear, and a band of approximately 5 kb should appear concomitant with duplex circle formation (Fig. 6A). In Fig. 6B at left, complete strand exchange in the presence of RecA alone appears to be inhibited, with the appearance of a faint 5-kb band in the restriction-cleavage assay even after 120 min. In contrast, after only 30 min in the coupled RecA + RecJ reaction (right panel), the 1.9-kb and 3.0-kb fragments are depleted and the 5-kb band appears, indicating complete heteroduplex formation. We do not believe that these product bands are the result of thermal branch migration during the cleavage reaction after the removal of the RecA protein, because as little as 1 bp of non-homology will inhibit this type of transfer(20) . Therefore, strand exchange must have progressed through the non-homologous region to obtain these products. We note that the overall speed of this reaction was slightly less than the previous experiment which employed a more active RecA preparation. These data suggest that RecJ can promote efficient strand exchange through a non-homologous region, especially once heteroduplex joint molecules have been formed.
Figure 6: Restriction cleavage assay for completion of exchange. Strand-exchange reactions were performed between M13mp18 circular ssDNA and linear dsDNA substrate with a nonhomologous 187-bp region from plasmid pBR322 inserted 2.1 kb from the 3`(-)-end of the linear duplex. A, diagram of restriction cleavage assay for heteroduplex formation. Cleavage of linear DNA with AlwNI and BglII yield three fragments. Cleavage of the final product of strand exchange, the circular heteroduplex molecule, yields 2 fragments, with the terminal fragments of the linear DNA joined in the circular molecule. This fragment also extrudes a ssDNA loop where the region of non-homology was present on the linear duplex substrate. B, restriction analysis of reactions with dsDNA linear substrate carrying internal non-homology. Time points of reactions with RecA alone are shown in the left panel and those with RecA + RecJ in the right panel. Completion of exchange is indicated by the appearance of a 5-kb band.
The addition of RecJ protein stimulated RecA-mediated strand
exchange between circular ssDNA and linear dsDNA, accelerating the
formation of the fully heteroduplex product. This stimulation required
that RecA had initiated transfer and that RecJ was present concurrent
with the exchange. Based on these data and the known ssDNA specificity
of RecJ, we propose a model (Fig. 1) where RecJ acts at a step
after initial formation of joint molecules. Once strand transfer
commences, a 5` single-strand end is produced, providing a substrate
for RecJ. RecJ is a processive nuclease and is able to degrade 75
nucleotides per s. ()Once RecJ binds to a displaced strand,
it may degrade the displaced DNA until it reaches the point where
RecA-mediated branch migration is occurring. The degradation of the
strand which competes for pairing with the transferred DNA strand may
therefore drive forward the unidirectional branch migration mediated by
RecA protein.
A similar mechanism has been proposed, in certain instances, for the stimulation of strand exchange by single-strand DNA binding proteins. In the T4 bacteriophage in vitro system, the ability of the UvsX protein to perform strand exchange is stimulated by the gene 32 protein, by either displacing UvsX from the invading strand or by binding to the displaced strand(21) . Likewise, RecA reactions at a low magnesium concentration under volume-exclusion conditions (22) show that SSB protein stimulates strand exchange at a postsynaptic step, presumably by binding to the displaced strand. The binding of these proteins to the displaced strand may aid the process of unwinding and, once the strand is displaced, may serve to sequester it and prevent it from pairing interactions. The RecJ-mediated degradation of the displaced strand may have a similar effect. It forces a unidirectional 5` to 3` branch migration and does not allow for RecA-mediated or thermal reversal of branch migration, driving the overall reaction forward.
This hypothesis may also explain the ability of RecJ to facilitate RecA-mediated branch migration through regions of non-homology. Under our reaction conditions, a nonhomologous region of 187 bp internal to the duplex substrate substantially inhibited the RecA strand-exchange reaction, but the addition of RecJ completely alleviated this block to transfer. This result supports the idea that the degradation of the displaced strand pushes the reaction forward, even through the non-homology, to product formation. The nonhomologous duplex DNA may become unpaired transiently by RecA or by thermal denaturation; RecJ degradation then ``traps'' this unwinding and, when the duplex DNA is unwound and digested sufficiently to reveal a homologous region, strand transfer is able to resume. In addition, RecJ did allow RecA to overcome a non-homology block at the 3`(-)-strand of the duplex, although incompletely. With such a block, even joint molecule formation is normally inhibited(17, 18) . In this latter case, some RecA-mediated or thermal denaturation of the nonhomologous end of the linear duplex may allow RecJ to digest past the point of non-homology. However, RecJ-bypass of the terminal block did not appear to be as efficient as that observed in the case of the internal non-homology block, where branch migration had been initiated by RecA and stalled at the non-homology. This appears to mirror the case of the rate stimulation above, where RecJ aided RecA effectively only after heteroduplex joints had been formed, most likely because branch migration facilitates unwinding.
RecJ degradation may free RecA of the requirement for continuous strand-pairing in genetic exchange and allow strand exchange to proceed with an imperfect RecA-ssDNA filament. In vivo, the RecA filament on ssDNA may be discontinuous. RecJ degradation may allow strand exchange to ``jump'' over the RecA-less regions, as it allows RecA in vitro to bypass regions of non-homology in branch migration. In support of this idea, we have also observed RecJ-mediated stimulation of the RecA strand-exchange reaction in the absence of SSB protein. In this instance, secondary structure in the ssDNA prevents complete RecA-ssDNA filament formation, and strand exchange is inefficient(14, 15) .
The enhancement of the process of RecA-mediated strand exchange by the RecJ exonuclease suggests a new role for exonucleases in genetic recombination. An implication of this role is that regions of heteroduplex DNA formed during genetic recombination may be less extensive in exonuclease-mutant strains. It may be that other single-strand specific exonucleases such as exonuclease I and exonuclease VII, also enhance the strand-transfer reaction in the Escherichia coli cell. The addition of exonuclease I, a 3`- to 5`-ssDNA exonuclease(23, 24) , or exonuclease VII, a ssDNA exonuclease with both 3` to 5` and 5` to 3` activities(25) , to the RecA-strand-transfer reaction has been shown previously to overcome a 3`(-)-non-homology block to the reaction and may change the polarity of strand exchange(26, 27) .