(Received for publication, April 2, 1997)
From the Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706
ATP hydrolysis (by RecA protein) fundamentally alters the properties of RecA protein-mediated DNA strand exchange reactions. ATP hydrolysis renders DNA strand exchange unidirectional, greatly increases the lengths of hybrid DNA created, permits the bypass of heterologous DNA insertions in one or both DNA substrates, and is absolutely required for exchange reactions involving four DNA strands. There are at least two viable models to explain how ATP hydrolysis is coupled to DNA strand exchange so as to bring about these effects. The first couples ATP hydrolysis to a redistribution of RecA monomers within a RecA filament. The second couples ATP hydrolysis to a facilitated rotation of the DNA substrates. The RecA monomer redistribution model makes the prediction that heterology bypass should not occur if the single-stranded DNA substrate is linear. The facilitated DNA rotation model predicts that RecA protein should promote the separation of paired DNA strands within a RecA filament if one of them is contiguous with a length of DNA being rotated about the filament exterior. Here, a facile bypass of heterologous insertions with linear DNA substrates is demonstrated, providing evidence against a role for RecA monomer redistribution in heterology bypass. In addition, we demonstrate that following a four-strand DNA exchange reaction, a distal segment of DNA hundreds of base pairs in length can be unwound in a nonreciprocal phase of the reaction, consistent with the direct coupling of an ATP hydrolytic motor to the proposed DNA rotation.
RecA protein plays an essential role in recombinational repair and homologous recombination in Escherichia coli. A DNA strand exchange reaction that can involve either three or four DNA strands has been widely used as a model for recombinational processes in vivo (1-3).
RecA protein-mediated DNA strand exchange occurs in several recognized phases. First, RecA protein binds to DNA1, typically either a circular ssDNA1 (for a three-strand reaction) or circular gapped duplex (four-strand reaction), forming a contiguous nucleoprotein filament. Next, a homologous linear duplex DNA (DNA2) is aligned with the ssDNA within the filament. Under typical conditions used in vitro, 1-2 kbp of DNA are then rapidly exchanged in the three-strand reaction after alignment. Finally, the nascent hybrid DNA product is extended in a slower, progressive reaction, which is able to bypass a variety of structural barriers. This leads ultimately to a nicked circular hybrid duplex DNA product along with a displaced linear single strand or partially duplex product.
The first three phases of DNA strand exchange, up to the formation of a
nascent region of hybrid DNA, require ATP binding to RecA protein but
not ATP hydrolysis. This has been demonstrated by the use of the ATP
analog ATPS (which is bound but not appreciably hydrolyzed by RecA)
or the RecA mutant protein K72R (which binds but does not hydrolyze
ATP) (4-11). In contrast, extension of the hybrid DNA in the final
phase requires ATP hydrolysis (9, 11, 12). ATP hydrolysis renders this
final phase of the strand exchange reaction unidirectional, with the
branch moving uniquely 5
3
relative to the single-stranded DNA in
DNA1 (9, 13, 14). ATP hydrolysis is also required for the bypass of
short heterologous insertions or other structural barriers in either DNA substrate (5, 6, 11) and for strand exchange reactions involving
four DNA strands (7, 11). Thus, ATP hydrolysis appears to confer
properties on DNA strand exchange that are important for
recombinational DNA repair (3, 15-17).
The mechanism by which ATP hydrolysis influences the last phase of DNA
strand exchange has not been elucidated. A number of early proposals
coupled strand exchange to disassembly of RecA filaments at or near the
migrating DNA branch, sometimes with reassembly on the displaced single
strand (13, 18, 19). ATP hydrolysis is required for the
end-dependent disassembly of RecA filaments, which has
recently been observed and characterized for filaments on both single-
and double-stranded DNA (20-22). The disassembly occurs from the end
opposite to that at which RecA protein is added during filament
assembly, proceeding 5
3
on single-stranded DNA (22), consistent
with the overall direction of the DNA strand exchange reaction.
However, DNA strand exchange requires no net disassembly of RecA
filaments, and RecA protein remains quantitatively bound to the hybrid
DNA product of DNA strand exchange after the reaction is complete under
optimal conditions in vitro (23, 24). In addition, the rate
of end-dependent filament disassembly is highly
pH-dependent, being negligible at pH 6 and relatively high
above pH 8, while the rate of RecA-mediated DNA strand exchange is
constant over the same pH range (20, 22). These and other results have
effectively eliminated models involving net disassembly of RecA
filaments as a required feature of the DNA strand exchange mechanism
(3).
Two additional models have been proposed that are generally consistent with observations published to date. The first, proposed by Kowalczykowski and colleagues (4, 8, 10), is based on the premise that RecA-mediated DNA strand exchange in the absence of ATP hydrolysis is limited only by discontinuities in the RecA filament. ATP hydrolysis would therefore be needed to redistribute RecA monomers to eliminate the discontinuities. An exchange of RecA monomers in the filament interior between free and bound forms has been observed when RecA protein is bound to duplex DNA (25) and also occurs during DNA strand exchange reactions (26). This fulfills a key requirement of the RecA monomer redistribution idea.
The second model involves a facilitated rotation of segments of the DNA substrates about each other, coupled to ATP hydrolysis (3, 11, 12, 16). This model neither requires nor precludes a limited exchange of RecA monomers between free and bound forms. Recent measurements of the rates of ATP hydrolysis and DNA branch movement during DNA strand exchange conform closely to predictions of the facilitated DNA rotation model (12).
These models make a number of additional and testable predictions. To
be successful, both models must explain the requirement for ATP
hydrolysis both in the bypass of heterologous DNA insertions and for
four-strand exchange reactions. As shown by Jwang and Radding (27), the
bypass of heterologous insertions involves the application of torsional
stress to unwind any heterologous insert in the duplex DNA. In the
facilitated DNA rotation model, the required unwinding is a direct and
predictable result of the rotation of one DNA substrate about the
other, coupled to an ATP-driven motor (3, 16, 28). In contrast, RecA
redistribution could in principle bring about unwinding of the
heterologous insertion indirectly (Fig. 1). The DNA within a RecA
filament is underwound by about 40% (29, 30). Upon dissociation of a
segment of RecA filament, the underwinding could be translated into
strand separation in the insertion, with up to eight bp separated for
every six RecA monomers dissociated, provided that free rotation of the filament was constrained to prevent spontaneous relaxation of the freed
DNA (Fig. 1). This condition might be met in a RecA filament formed on
circular DNA where there were no free nucleoprotein filament ends.
However, if the RecA filament were formed on linear ssDNA, the RecA
redistribution model predicts that insertion bypass should not occur.
This is because any DNA underwinding liberated by RecA dissociation
would be immediately relaxed by simple and unconstrained rotation about
the long axis of the filament. Insertion bypass by facilitated DNA
rotation, in contrast, should occur whether the ssDNA substrate is
linear or circular.
DNA strand exchange involving four strands presents both models with a
different set of challenges. Under conditions in which ATP is not
hydrolyzed, strand exchange can be initiated by pairing DNA2 within the
single strand gap of DNA1, where the reaction involves only three of
the four strands. However, the reaction does not proceed beyond the gap
unless ATP is hydrolyzed (7, 11). In any strand exchange reaction, the
ends of DNA2 are often referred to as proximal and distal, reflecting
the ends at which a unidirectional strand exchange begins and ends,
respectively; we use this terminology to facilitate the continuing
discussion. The proximal end of linear duplex DNA2 generally overlaps
the ssDNA gap of DNA1, and at the distal end it is either coincident with or overlaps the gap of DNA1 (Fig. 2A). At the proximal
end of DNA2, the overlap reflects the requirement to initiate even a
four-strand reaction in a three-stranded region (31, 32). The distal
end of DNA2 must also be at least coincident with the end of the duplex
in DNA1 so as to permit a reciprocal exchange throughout the
four-stranded part of the reaction (Fig. 2A). If the duplex
portion of DNA1 extends beyond the distal end of DNA2, the strand
exchange reaction might be expected to halt where the reciprocal
reaction ends. Completion of the reaction would require a helicase-like
separation of the strands in the distal duplex extension in DNA1 (Fig.
2B), and RecA has no significant intrinsic helicase activity
(33). However, if RecA promotes an ATP-dependent DNA
rotation, this activity might produce an unraveling of the duplex
extension by means of the torsional stress generated in the DNA by the
ATPase motor.
This report describes our tests of the predictions presented above.
E. coli RecA protein was
purified to homogeneity and stored as described previously (34).
E. coli ssDNA-binding protein (SSB) was purified to
homogeneity as described (35), except that an additional step utilizing
DEAE-Sepharose chromatography was included to ensure removal of
single-stranded exonucleases. The RecA protein and SSB concentrations
were determined by absorbance at 280 nm, using extinction coefficients
of 280 = 0.59 A280
mg
1 ml (36) and
280 = 1.5 A280 mg
1 ml (35), respectively.
RecA and SSB preparations were free of detectable endo- and exonuclease
activities on double- or single-stranded DNA. Restriction
endonucleases, Vent polymerase, T4 polynucleotide kinase, and
-agarase I were purchased from New England Biolabs. Tris buffer was
purchased from Fisher. DEAE-Sepharose was purchased from Pharmacia
Biotech Inc. Pyruvate kinase, phosphoenolpyruvate, creatine
phosphokinase, phosphocreatine, and ATP were purchased from Sigma.
Ultrapure dATP was from Pharmacia. Low melting agarose was purchased
from FMC and Sigma. Microcon-10 microconcentrators were purchased from
Amicon. 4
-amino-4,5
,8-trimethyl-psoralen (AMT) was purchased from
Calbiochem.
Duplex and ssDNA substrates were derived from
bacteriophage M13mp8 (37). Bacteriophages M13mp8.52 and M13mp8.121
were constructed by replacing the 30-bp
EcoRI-HindIII fragment of bacteriophage M13mp8
with 52- or 121-bp heterologous DNA fragments as described (38).
Bacteriophage M13mp8.198 is bacteriophage M13mp8 with 198 bp
(RsaI-RsaI fragment from the E. coli
galT gene) inserted into the SmaI site (21).
Bacteriophage M13mp8.1037 is bacteriophage M13mp8 with 1037 bp
(EcoRV-EcoRV fragment from the E. coli
galT gene) inserted into the SmaI site (32).
Supercoiled circular duplex DNA and circular single-stranded DNA from
bacteriophage M13mp8 and its derivatives were prepared using methods
described previously (37, 39, 40). The concentrations of dsDNA and ssDNA stock solutions were determined by absorbance at 260 nm, using 50 and 36 µg ml1
A260
1, respectively, as
conversion factors. DNA concentrations are expressed in terms of total
nucleotides.
Complete digestion of supercoiled M13mp8 derivatives with appropriate
restriction enzymes resulted in linear duplex DNA substrates. After
digestion, residual protein was removed by sequential 1:1 extractions
with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl
alcohol (24:1) followed by ethanol precipitation. When necessary,
linear duplex DNA fragments were purified by separation in low melting
agarose gels and removed from the gels with -agarase according to
manufacturer's instructions.
Full-length linear ssDNA substrates were generated by digestion with
AlwNI after 18 base oligonucleotides complementary to the
AlwNI site were annealed to the ssDNA. After digestion,
residual protein was removed by 1:1 extraction with
phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl
alcohol (24:1). Excess oligonucleotides were removed by washing the
solution through a microconcentrator. The substrates were concentrated
by ethanol precipitation. After cleavage, the annealed oligonucleotide
remnants paired to the very ends of the linearized ssDNA are quite
short and generally lost during the subsequent procedures. We did not confirm their loss in the current study, since none of the experiments involved pairing or strand exchange at any location near the ends where
these oligonucleotides may have been paired. Linear ssDNA fragments
were similarly generated by digesting ssDNA, annealed to appropriate
18-base oligonucleotides, with PacI and BglII. The cleaved DNA was isolated from preparative low melting agarose gel
using -agarase, according to the manufacturer's instructions. The
fragments were extracted 1:1 with phenol/chloroform/isoamyl alcohol
(25:24:1) and chloroform/isoamyl alcohol (24:1). The fragments were
concentrated by isopropyl alcohol precipitation.
Gapped duplex DNA molecules were prepared by large scale RecA protein-mediated strand exchange reactions between circular ssDNA and linear duplex DNA molecules.
Circular duplex DNA with a defined gap, GD2038, was
prepared from circular M13mp8.1037 ssDNA and
AvaII-BglII-cut duplex DNA molecules of M13.mp8.
The AvaII-BglII-cut duplex molecules were purified from an agarose gel with -agarase, according to the manufacturer's instructions.
Other gapped duplexes, GS1, GS2,
GS3, and GS4, were prepared from circular
M13mp8.198 ssDNA and linear, PCR-generated DNA molecules. Linear duplex
DNA molecules were prepared by PCR using a Perkin-Elmer DNA thermal
cycler. All PCR reactions were performed in a standard reaction buffer
containing 10 mM KCl, 20 mM Tris-Cl (pH 8.8 at
25 °C) 10 mM
(NH4)2SO4, 2 mM
MgSO4, 0.1% Triton X-100, 200 µM
concentration of each nucleotide (dATP, dTTP, dCTP, and dGTP), 250 nM concentration (in total oligonucleotides) of each primer, 20 units ml1 Vent DNA polymerase, and 2 µg
ml
1 M13mp8.198 DNA (linearized by BamHI). The
total reaction volume was 100 µl. Denaturation, annealing, and
polymerization temperatures were 94, 55, and 72 °C, respectively,
and amplification was carried out for 25 cycles. The PCR products were
separated on a 1% agarose gel, and the amplified fragment was excised
and purified from low melting agarose by a published procedure (41).
The DNA was ethanol-precipitated and resuspended in Tris/Cl-EDTA. Some
PCR products were 5
-end-labeled as described (41).
The primers used for the 1947-bp fragment 1 were
5-GAAATGAATAATTCGCCTCTGCG-3
and 5
-GGGAATTCGTAATCATGGTCAT-3
,
yielding a fragment corresponding to positions 4292-6238 on the
M13mp8.198 map. The G + C content of the 1947-bp fragment was 45%. The
183-bp fragment 2 used primers 5
-ACCCATGGGTGCAGGTTTTTGAA-3
and
5
-GCTCGCGCTGAACATAATCCAC-3
, yielding a fragment corresponding to
positions 6239-6421 on the M13mp8.198 map (G + C content = 53%).
The 2130-bp fragment 3 used the first and last of these four primers,
yielding a fragment corresponding to positions 4292-6421 on the
M13mp8.198 map (G + C = 45%).
The linear duplex and circular ssDNA molecules were combined in large
scale RecA-mediated three-strand exchange reactions to generate gapped
duplex DNA molecules. Reactions contained 7 µM RecA
protein, 2 µM SSB, 3 mM ATP, and 21 µM circular single-stranded DNA. The reactions to
generate gapped duplex substrates also contained one or more linear
duplex DNA molecules: GD2038, 16 µM M13mp8 DNA cut with AvaII and BglII; GS1,
5.5 µM PCR fragment 1; GS2, 6.0 µM PCR fragment 3; GS3, 11 µM
PCR fragment 1 plus 2.1 µM 5-end-labeled PCR fragment 2;
GS4, 2.1 µM 5
end-labeled PCR fragment 2. The reaction conditions were otherwise as described below, except that
reaction volumes ranged from 0.6 to 1.4 ml. After 2 h of incubation, the DNAs were concentrated in Microcon-10
microconcentrators, and the products were separated on a 1.0% agarose
gel. Gapped duplex DNA molecules were purified from the gel with
-agarase, according to the manufacturer's instructions.
Reactions between
linear ssDNA and linear dsDNA were carried out at 37 °C in a
standard reaction buffer containing 25 mM Tris acetate
(80% cation), 10 mM magnesium acetate, 3 mM
potassium glutamate, 1 mM dithiothreitol, 5% glycerol
(w/v), and a dATP-regenerating system (20 units ml1
pyruvate kinase and 10 mM phosphoenolpyruvate). The pH of
the reaction mixture was 7.0 after the addition of all components except proteins and DNA (storage buffers were substituted for protein
and DNA solutions in the pH measurement). Reaction volumes were 55 µl, and concentrations of DNA and proteins reported below are the
final concentrations after the addition of all components. Single-stranded DNA (20 µM) and duplex DNA were incubated
with RecA protein (10 µM) for 10 min before dATP (3 mM) and SSB (2 µM) were added as a mixture to
start the reactions. Duplex DNA and ssDNA molecules were present in a
1:1 ratio.
Reactions between gapped circular duplex DNA and linear dsDNA were
carried at 37 °C in a standard reaction buffer containing 25 mM Tris acetate (80% cation, pH 7.5), 10 mM
magnesium acetate, 3 mM potassium glutamate, 1 mM dithiothreitol, 5% glycerol, and an ATP-regenerating
system (10 units ml1 creatine phosphokinase and 12 mM phosphocreatine). The concentrations of DNA and proteins
reported here are the final concentrations after the addition of all
components. ATP and SSB were added to start strand exchange reactions
after the incubation of linear duplex and gapped duplex DNA with RecA
protein at 37 °C for 10 min.
Aliquots (12 µl) of strand exchange reactions
described above were removed at 0, 20, 40, and 90 min, and the
reactions were stopped by the addition of 6 µl of gel-loading buffer
(25% glycerol, 15 mM EDTA, 0.025% bromphenol blue, 5%
sodium dodecyl sulfate). These aliquots were stored on ice until the
last time point was taken. Samples were electrophoresed overnight in an
0.8% agarose gel at 2-2.5 V cm1. In some cases, gels
were dried and bands were visualized by autoradiography. To prevent the
loss of small DNA fragments during gel drying, gels were dried on
DEAE-cellulose paper. The DEAE-cellulose paper was prepared by soaking
for 5 min in 10 mM EDTA (pH 8.0), soaking for 5 min in 1 N NaOH, and washing six times with double distilled
H2O (41).
Samples for electron microscopy were
obtained by either elution of DNA product bands from agarose gels or by
spreading the entire strand exchange reaction mixture as noted. Elution
of DNA from 0.7% agarose gels was carried out by adding 90 mM Tris borate and 2 mM EDTA to excised and
chopped gel slices, followed by incubation for 10 min at 37 °C and
overnight at 4 °C. The samples were spun for 10 min in a
microcentrifuge, and the supernatants were collected. The
centrifugation was repeated. Samples from entire strand exchange reaction mixtures were cross-linked with AMT prior to spreading. Aliquots (15 µl) of the strand exchange reactions at the 120-min time
point were mixed with AMT (30 mg ml1, final
concentration), incubated at 25 °C for 3 min, and irradiated with
long wave UV light for 4 min at 25 °C (42). Cross-linked samples
were incubated with proteinase K (1 mg ml
1 final
concentration) and sodium dodecyl sulfate (0.9% final) for 60 min at
37 °C. All samples were dialyzed into 20 mM NaCl and 5 mM EDTA overnight at 25 °C on Millipore type VM
(0.05-mm) filters and were then spread as described previously (43).
Photography and measurements of the DNA molecules were performed as
described (44).
There were two sets of experiments. In the first we compared DNA strand exchange reactions between either linear or circular ssDNA and linear duplex DNA to test a mechanism for the bypass of heterologous DNA insertions outlined in Fig. 1. The second was an attempt to test the facilitated DNA rotation model using four-strand DNA exchange reactions outlined in Fig. 2.
Testing RecA Monomer Redistribution: Heterologous DNA Insertions Were Bypassed Even When the ssDNA Substrate Was LinearA key
issue in the experimental design was the stability of the filaments. At
least one report (45) and our own experience have indicated that DNA
strand exchange reactions involving linear ssDNA substrates are less
efficient than those with circular ssDNA. This reflects filament
instability in the form of a recently characterized end-dependent disassembly of RecA filaments from ssDNA in
the presence of SSB that proceeds 5
3
(22).
End-dependent RecA filament disassembly is blocked at pH
values below 6.5 or if dATP replaces ATP as a nucleotide cofactor or if
the RecO and RecR proteins are present in the reaction mixture (22).
From an experimental standpoint, the simplest way to stabilize the RecA
filament on linear ssDNA is to use dATP instead of ATP. The nucleotide
dATP is hydrolyzed by RecA protein at rates about 20% higher than
observed with ATP yet has a general stabilizing effect on RecA
filaments and supports DNA strand exchange reactions somewhat better
than ATP (22, 46). The in vivo significance of the effects
of dATP are not clear.
The reactions of circular or linear ssDNA with 1.8-kbp duplex DNA
fragments, some containing a 52- or 121-bp heterologous insert (Fig.
3, substrates A plus C-E), were monitored on
an agarose gel (Fig. 4). The expected product of
complete strand exchange contains 1.8 kbp of hybrid duplex DNA (with a
bubble corresponding to the insert sequences) and 5.4 kbp of ssDNA.
Where linear ssDNA was used, the product has 2.9 and 2.5 kb of ssDNA,
respectively, on the proximal and distal sides of the 1.8-kbp region of
hybrid duplex DNA. In all six reactions, production of hybrid duplex product was readily observed. In general, the reactions with the linear
ssDNA substrates appeared to be very slightly slower than the
corresponding reactions involving circular ssDNA, but the reaction end
points were very similar. All of the reactions shown in Fig. 4 were
repeated at least three times with consistent results. Reaction
efficiency decreased as the length of the heterologous insert
increased, consistent with published results (5, 6, 11, 19, 47). In an
experiment similar to that of Fig. 4, a 198-bp insert was not bypassed
with either circular or linear ssDNA (data not shown).
Bypass is also observed when the 52-bp heterologous insertion is moved
from the dsDNA to the linear ssDNA substrate (Fig. 3, substrates
B plus D). The reactions were again monitored on an agarose gel
(Fig. 5). The expected gapped duplex product was generated in each reaction. This bypass of heterology in the ssDNA substrate was somewhat less efficient than bypass of heterology in the
duplex substrate for both circular and linear RecA nucleoprotein filaments, consistent with previous observations (6).
A RecA filament formed on linear ssDNA need only rotate about its long
axis to relieve any torsional stress introduced in the DNA by
dissociation of interior monomers. The molecular mass of a contiguous
RecA filament formed on the ssDNA substrates used in Figs. 4 and 5 is
in excess of 90,000,000 daltons. To address any possibility that the
mass itself is a barrier to free rotation, strand exchange reactions
were conducted using ssDNA substrates that had been shortened by more
than 60%, decreasing the mass of the filaments formed on them by a
like amount (Fig. 3F). Where applicable, the 52-bp insertion
was in the duplex substrate (Fig. 3H). With the shorter
linear ssDNA substrate, strand exchange reactions generated the
expected product whether the 52-bp insertion was present or not (Fig.
6). Both the rates and the end points of the reactions
were comparable with the reactions involving the much longer linear
ssDNA substrates in Fig. 4.
Testing Facilitated DNA Rotation: RecA Protein Can Unwind a Distal Segment of Duplex DNA as the Final Stage of a Four-strand Exchange Reaction
The question introduced in Fig. 2 was addressed in two experiments. The first was set up to determine if a complete DNA strand exchange could occur if the duplex region of the gapped duplex substrate extended beyond the end of DNA2, as in Fig. 2B. The experiment also addressed the effect of the length of the duplex extension in DNA1 on reaction efficiency. The second experiment was carried out to determine if severing the covalent link between the duplex extension and the remainder of the DNA strand (as in Fig. 2C) affected the reaction.
The substrates used in the first experiment, a gapped duplex substrate
and a series of linear duplex substrates shortened progressively at the
distal end, are illustrated in Fig. 7. In a standard
RecA protein-mediated four-strand exchange reaction, the linear duplex
DNA overlaps the single strand gap on the proximal end. The distal end
of the linear duplex DNA substrate also overlaps the single-stranded
region of the gapped duplex or extends at least up to the duplex/single
strand junction. After strand exchange enters the duplex region of the
gapped duplex to convert a three-strand exchange to a four-strand
exchange, the exchange reaction remains reciprocal through the end of
the reaction. Gapped duplex and linear products accumulate as the
reaction progresses (Fig. 8).
The effect of shortening the distal end of the linear duplex DNA substrate on a four-strand exchange reaction is shown in Fig. 8. In a reaction between a gapped duplex and a linear duplex lacking 88 bp from the distal end, separate linear and gapped duplex products formed (Fig. 8). The resolution of the two products indicated that the 88-bp on the distal end of the gapped duplex substrate were unwound in the last stage of the reaction. Products were evident within 40 min, although this reaction was less efficient than the reaction involving a full-length linear substrate (Fig. 8). As the length of the duplex region requiring unwinding increased, the efficiency of strand-exchange decreased somewhat further. However, linear duplex substrates lacking 199, 302, and 835 bp from the distal end all reacted to form detectable discrete products (Fig. 8). No unwinding was observed when the length of the distal segment to be unwound was increased to 3722 bp (data not shown).
We investigated the possibility that the relatively low level of
completed products in the nonreciprocal exchange reactions in Fig. 8
was due to nuclease contamination. An endonuclease could convert
molecules in which strand exchange had stalled at the distal extension
in the duplex of DNA1 to molecules migrating like products if it
cleaved off the DNA branch. This would generate products with a ssDNA
gap shorter than those expected if the products were generated by
unwinding the distal extension, since the distal extension would still
be present. Alternatively, an exonuclease could act on the interrupted
strand in the gapped circle, creating gaps of variable size and
sometimes removing the DNA branch. To determine the length of the ssDNA
gap in the products, the product bands from some reactions were excised
from the gel, and the DNA was examined by electron microscopy.
Representative gapped duplex products from strand exchanges with distal
unwinding of 835 and 88 bp are shown in Fig. 9,
panels A and B and panels C and
D, respectively. The length of the ssDNA gap in products
such as those in Fig. 9, A and B, was measured
and found to be consistent with complete unwinding of the 835-bp distal
region. The measured length of the gap was 1770 ± 90 nucleotides
(nine molecules measured), compared with a gap of 1613 nucleotides that
would be generated by unwinding of the distal extension. No molecules
were observed with a gap size approximating the 778-nucleotide gap
expected if a nuclease simply removed the DNA branch. When the entire
835-bp unwinding reaction mixture was spread for electron microscopy, the predominant species was found to be a branched intermediate, apparently stalled at the junction with the distal duplex extension in
DNA1 (Fig. 9, E and F). This is consistent with
the results in Fig. 8. There was no evidence of unusual variability in
gap size of the sort that might result from a contaminating
exonuclease, either in the complete reaction mixture or in the gapped
DNA in the product band. We conclude that the products generated in the reactions requiring nonreciprocal exchange resulted from unwinding of
the distal segment rather than from nuclease action.
Similar EM results were obtained in an examination of products such as those in Fig. 9, C and D. The measured gap size (970 ± 70 nucleotides; 14 molecules measured) is more consistent with the gap expected if the distal 88 bp is unwound (866 nucleotides) than with the gap expected if the branch is removed by nuclease action (778 nucleotides). However, the differences in gap sizes are smaller and less meaningful in this instance than they are when unwinding of 835 bp is required.
The yield of discrete products generated in these reactions depended
upon the concentrations of magnesium ion, linear dsDNA substrate, and
SSB as well as the length of time the reactions were followed. The
reactions were optimal at magnesium acetate concentrations of between 6 and 12 mM (data not shown). A stronger reaction than that
observed in Fig. 8 could be observed, at least for the shorter distal
segments, if the ratios of SSB and linear dsDNA to other reaction
components was increased, and the reaction time course was extended
(Fig. 10). The effect of SSB was especially important
(data not shown). Doubling the SSB:DNA substrate ratio (as was done in
Fig. 10 relative to Fig. 8) greatly increased the yield of products at
all time points, despite the fact that the SSB concentration under
either set of conditions is greatly in excess relative to the amount of
single-stranded DNA available for binding. We hypothesize that the
excess SSB is required to ensure that the separated complementary
strands do not reanneal. Adding the excess SSB prior to RecA protein,
which should inhibit the nucleation of RecA filaments, had a
substantial inhibitory effect on the reaction (Fig. 10).
As a final control, we tested the possibility that simple instability of the branched reaction intermediates could contribute to the yield of products. A set of reactions was carried out with the DNA and RecA protein concentrations of Fig. 10 but with a wide range of magnesium acetate and SSB concentrations, encompassing the optimum for both variables. Incubation at 37 °C for 18 h, enough time for the ATP regeneration system to be long depleted, did not increase the yield of products over that observed at 4 h but instead decreased it somewhat (data not shown). The yield of branched DNA intermediates did not decrease. We conclude that the intermediates do not undergo significant breakdown under these conditions and that spontaneous reannealing of the products may occur and lead to an underestimation of overall reaction efficiency.
Unwinding of the Distal Duplex DNA Segment Requires a Covalent Link to DNA Undergoing a Reciprocal Strand ExchangeThe nonreciprocal
DNA unwinding could represent a cryptic helicase activity of the RecA
protein monomers bound to the DNA being unwound or a contaminating
helicase, perhaps activated in some way by the strand exchange process.
Alternatively, if the four-strand DNA exchange is brought about by
facilitated DNA rotation, the distal segment might be unwound
indirectly as a result of the torsional stress generated by the
rotation of the DNA strands upstream of it. To distinguish between
these possibilities, a gapped duplex substrate was constructed with a
nick on the exchanged strand, separating the regions of reciprocal and
nonreciprocal exchange. The nick would preclude the transmission of any
torsional stress generated in the reciprocal strand exchange reaction
into the distal duplex DNA segment. It should not affect unwinding of
the duplex segment if unwinding is mediated directly by the RecA
protein that is bound to it. The substrates for this series of
experiments are illustrated in Fig. 11. The different
gapped duplexes are designated GS1-GS4.
In a fully reciprocal four-strand exchange reaction, with
GS1, the expected products were generated (Fig.
12A, reaction 1). Next, a strand
exchange reaction was conducted with a gapped duplex substrate
(GS2) containing a segment of duplex DNA extending 183 bp
beyond the distal end of the linear duplex. As expected from the
results in Fig. 8, the efficiency of the overall strand exchange is
reduced by the requirement that the 183-bp segment be unwound. However,
the generation of discrete products indicated that RecA protein can
unwind this distal duplex segment without reciprocal exchange (Fig.
12A, reaction 2). When the distal duplex segment was separated from the remainder of its strand in the gapped duplex substrate (GS3), the efficiency of the subsequent
four-strand exchange reaction was improved (Fig. 12A,
reaction 3). A three-strand exchange reaction also proceeds
efficiently when the distal end of the linear duplex substrate does not
overlap the same 183 bp representing the only duplex region in the
gapped duplex GS4 (Fig. 12A, reaction
4).
The fate of the 183-nucleotide DNA strand cannot be ascertained in reactions 3 and 4 of Fig. 12A. These reactions were therefore repeated, except that the 183-bp fragment of the gapped duplex substrate included a radiolabel to facilitate product identification (Fig. 12B). In the reaction corresponding to Fig. 12A, reaction 3, only the substrate and the gapped duplex product was radiolabeled. No free 183-bp fragment was observed (Fig. 12B, reaction 3). This indicates that the 183-bp distal duplex segment was not unwound when it was separated by a nick from the region of reciprocal exchange. The result was the same when the strand exchange reaction occurring proximal to the 183-bp duplex segment was limited to three strands Fig. 12A, reaction 4, and 12B, reaction 4). The 183-bp fragment was not unwound under any circumstances unless it was covalently linked to a strand involved in a reciprocal four-strand exchange reaction proximal to it.
Additional control reactions were conducted to further clarify the conditions required to separate the 183-bp fragment from the gapped duplex substrate. Under the same reaction conditions, but without a linear duplex substrate, the radiolabeled 183-bp fragment remained part of the gapped duplex when RecA protein was bound to it under otherwise identical reaction conditions (Fig. 12B, reaction C1). In addition, under the same conditions, but without linear duplex substrate, RecA protein, and SSB, the 183-bp fragment remained part of the gapped duplex (Fig. 12B, reaction C2), demonstrating that the substrate itself has no inherent instability. These results indicate that the unwinding of the nonreciprocal region was not caused by a direct, helicase-like, unwinding of the 183 bp.
Two sets of experiments with quite different experimental rationales are described in this report. They are unified only in that they both address the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. The first set of experiments demonstrates that the RecA redistribution model cannot explain the bypass of heterologous DNA insertions, one of the key effects of ATP hydrolysis. The second set of experiments presents a reaction that is readily explained only by a facilitated DNA rotation process. A coupling of ATP hydrolysis to rotation of segments of one DNA substrate about the other (as illustrated in detail elsewhere (3, 11, 26)) would expand the constellation of established RecA protein functions to include the role of the molecular motor.
The bypass of heterologous insertions during RecA protein-mediated DNA strand exchange is affected little when the ssDNA substrate is converted from a circular to a linear form. Insertions of at least 121 bp are bypassed with good efficiency. Good heterology bypass is also mediated by relatively short RecA filaments formed on linear ssDNA that is 2800 bases in length. These results provide a simple argument that heterology bypass does not occur by means of RecA monomer redistribution (as in Fig. 1). In a linear RecA nucleoprotein filament, any DNA underwinding uncovered by RecA dissociation should be readily relaxed by simple rotation of the nucleoprotein filament about its long axis. Since the DNA within a RecA filament is underwound by nearly 40% when ATP is being hydrolyzed (30), the release of 18 bp by dissociation of six RecA monomers could in principle be translated into strand separation over a stretch of 8 bp if filament rotation were constrained. The unwinding and bypass of a 52-bp insertion would therefore require the dissociation of a stretch of at least 40 RecA monomers, vacating 120 bp total, assuming that all of the released underwinding was translated into strand separation precisely at the heterologous insertion. The bypass of a 121-bp insertion would require the dissociation of over 90 RecA monomers, vacating over 270 bp of DNA. Rotation of a linear filament at its free ends would relax any DNA released by RecA dissociation, preventing the translation of underwinding into strand separation. The linear filaments used in this study should not promote the observed bypass of heterologous insertions if ATP hydrolysis serves only to redistribute RecA monomers.
In addition, there is no evidence that filament gaps of the required
size are ever created in the interior of a RecA filament. Filament
assembly processes (filament extension on the end corresponding to that
nearest the 3-end of a ssDNA lattice) are very rapid on either ssDNA
or dsDNA when a primer in the form of an appropriate filament end is
present (3, 48). Disassembly at the opposite end of the filament is
generally much slower (3, 20-22, 48). Any significant gap in the
filament interior should be quickly filled. To augment this point, we
note that doubling the concentration of RecA protein (to twice the
amount needed to saturate the ssDNA) does not inhibit the bypass of a
52-bp heterologous DNA insertion,2 as it
might if partial disassembly of the filament were a requirement for
bypass.
The heterology bypass with linear DNA substrates is readily explained
by the facilitated DNA rotation model (3, 11, 12, 16). A second set of
experiments addresses the facilitated DNA rotation model more directly.
RecA protein promotes the complete unwinding of hundreds of base pairs
of duplex DNA as the final stage of an otherwise reciprocal four-strand
exchange reaction. The unwinding occurs only if both strands are
contiguous with DNA strands directly involved in the four-strand
exchange portion of the reaction. The unwinding mechanism therefore
involves an indirect application of torsional stress, as opposed to a
direct helicase-like melting of the DNA by bound RecA. The experiments in Figs. 7, 8, 9, 10, 11, 12 were inspired in part by the work of Jwang and Radding
(27), and our results are consistent with their work. These results are
also readily explained by facilitated DNA rotation of the DNA
substrates coupled to ATP hydrolysis (16). Unwinding of the duplex DNA
segments would occur as a byproduct of rotating the hybrid DNA
segments, as illustrated in Fig. 13. This mechanism
requires no disassembly of RecA filaments to bring about the
unwinding.
The bypass of heterologous insertions during three-strand exchanges displays a complete requirement for ATP hydrolysis (6). We infer that the unwinding of the duplex segments documented in the present study also requires ATP hydrolysis. We cannot directly test this, however, because the four-strand exchange that must precede the duplex unwinding also requires ATP hydrolysis (7, 11).
This work must be considered in the context of a range of recent findings. When ATP is not hydrolyzed by RecA protein (e.g. when the RecA K72R mutant protein is used in place of the wild type), some DNA strand exchange occurs, but it is limited in extent (4, 8-11) and exhibits other limitations enumerated in the Introduction. The RecA redistribution model posits that DNA strand exchange without ATP hydrolysis is limited only by discontinuities in the RecA filament and that ATP hydrolysis is needed only for RecA monomer redistribution to repair the discontinuities. The exchange of RecA monomers between free and bound forms that is required by this model has been observed during DNA strand exchange (26). However, DNA strand exchange is blocked in mixed filaments of wild type and K72R mutant protein under conditions where a facile exchange of RecA monomers between free and bound forms is observed (26). Also, the addition of excess RecA K72R protein to reactions (sufficient to fill in any filament discontinuities) does not overcome the barrier to more extensive strand exchange. RecA redistribution is therefore not sufficient in itself to overcome the limited extent of strand exchange seen when ATP is not hydrolyzed. We demonstrate here that RecA redistribution also cannot account for the requirement for ATP hydrolysis in the bypass of heterologous DNA insertions. No effort has been made to explain how RecA redistribution might explain the requirement for ATP hydrolysis in four-strand exchange reactions. The role of RecA monomer exchange in filament interiors (25) in the mechanism of DNA strand exchange, if any, remains to be elucidated.
The facilitated DNA rotation model replaces the discontinuous filament with a discontinuous DNA pairing intermediate. Segments in which the duplex DNA substrate is interwound with the ssDNA within the filament alternate with segments in which the duplex DNA is looped outside the filament (3, 11, 26). Since homologous DNA pairing occurs in the segments contained within the RecA filament, this idea does not conflict with any current proposal for the alignment of three DNA strands by RecA protein. In the absence of ATP hydrolysis, the DNA substrates are paired, and strand exchange is promoted by binding energy within the filament groove, but it can proceed only up to the first external loop. We point out that discontinuous DNA pairing intermediates of the required type are formed under conditions where ATP hydrolysis (and therefore extensive DNA strand exchange) is blocked and can be observed directly by electron microscopy (11, 49). Beginning with these intermediates, facilitated DNA rotation provides a comprehensive explanation for the observed effects of ATP hydrolysis on DNA strand exchange (3, 16) as well as for the quantitative relationship between the rates of branch movement and ATP hydrolysis (12).
Traditionally, bacterial recombination has been explored within a paradigm defined by conjugational recombination (17, 50). However, a molecular motor function for the RecA ATPase is best rationalized within a paradigm focused on recombinational DNA repair, where unidirectionality and the capacity to bypass structural barriers in the DNA would be critical features of DNA strand exchange (15, 16). The ATP binding site represents the most conserved portion of the RecA protein structure (3), yet ATP hydrolysis appears to have no role in the fundamental process of DNA pairing. DNA pairing and significant DNA strand exchange is promoted by a RecA mutant protein (K72R) that binds but does not hydrolyze ATP (8, 11). However, in vivo, this same mutation results in a RecA null phenotype (51). At least one other RecA mutant protein, with partially compromised DNA binding and ATP hydrolytic functions, will function adequately in conjugational recombination in vivo but will not function in recombinational DNA repair (52). These and other results (3, 15) indicate that recombination leading to repair is an inherently more demanding process than recombination during conjugation and that at least some of the molecular features of RecA protein evolved specifically to address the exigencies of repair. If the biochemistry of RecA protein and other bacterial Rec proteins is to be understood in all its complexity, we believe that the paradigm within which that understanding is sought should be shifted from conjugational recombination to recombinational DNA repair.
We thank Jong-Il Kim for carrying out some of the early experiments leading to the work described in this paper. We also thank Stephen Kowalczykowski for communicating, in 1994, ideas upon which the model in Fig. 1 was built.