(Received for publication, February 9, 1995; and in revised form, December 23, 1995)
From the
Replacement of lysine 72 in RecA protein with arginine produces
a mutant protein that binds but does not hydrolyze ATP. The protein
nevertheless promotes DNA strand exchange (Rehrauer, W. M., and
Kowalczykowski, S. C.(1993) J. Biol. Chem. 268,
1292-1297). With RecA K72R protein, the formation of the hybrid
DNA product of strand exchange is greatly affected by the concentration
of Mg in ways that reflect the concentration of a
Mg
dATP complex. When Mg
is present at
concentrations just sufficient to form the Mg
dATP complex,
substantial generation of completed product hybrid DNAs over 7 kilobase
pairs in length is observed (albeit slowly). Higher levels of
Mg
are required for optimal uptake of substrate
duplex DNA into the nucleoprotein filament, indicating that the
formation of joint molecules is facilitated by Mg
levels that inhibit the subsequent migration of a DNA branch. We
also show that the strand exchange reaction promoted by RecA K72R,
regardless of the Mg
concentration, is bidirectional
and incapable of bypassing structural barriers in the DNA or
accommodating four DNA strands. The reaction exhibits the same
limitations as that promoted by wild type RecA protein in the presence
of adenosine 5`-O-(3-thio)triphosphate. The Mg
effects, the limitations of RecA-mediated DNA strand exchange in
the absence of ATP hydrolysis, and unusual DNA structures observed by
electron microscopy in some experiments, are interpreted in the context
of a model in which a fast phase of DNA strand exchange produces a
discontinuous three-stranded DNA pairing intermediate, followed by a
slow phase in which the discontinuities are resolved. The mutant
protein also facilitates the autocatalytic cleavage of the LexA
repressor, but at a reduced rate.
The RecA protein of Escherichia coli is a 352-amino acid polypeptide chain with a predicted molecular weight of 37,842. The protein is found in all bacteria and is critical to the processes of recombinational DNA repair, homologous recombination, induction of the SOS response to DNA damage, SOS mutagenesis, and the partitioning of chromosomes at cell division (Clark and Sandler, 1994; Cox, 1994; Kowalczykowski et al., 1994; Stasiak and Egelman, 1994; Livneh et al., 1993; West, 1992; Roca and Cox, 1990).
In vitro, RecA protein promotes a set of DNA strand exchange reactions that mimics its presumed role in recombination and recombinational DNA repair. The reactions can involve either three or four DNA strands (Fig. 1). RecA first forms a nucleoprotein filament on the single-stranded or gapped DNA substrate. This DNA is then aligned with a homologous linear duplex DNA. A strand switch then occurs within the filament producing a nascent region of hybrid DNA, which is extended to generate the products shown. In a normal reaction, strand exchange proceeds unidirectionally, 5` to 3` relative to the single-stranded DNA (or the single-stranded region of the gapped DNA) to which the RecA first binds. The reaction also proceeds past a variety of structural barriers in the DNA substrates.
Figure 1: RecA protein-mediated DNA strand exchange reactions. Typical reactions involving three and four DNA strands are shown.
RecA protein
is a DNA-dependent ATPase, with a monomer k of
30 min
when bound to ssDNA. (
)ATP is
hydrolyzed uniformly throughout the nucleoprotein filament (Brenner et al., 1987). When a homologous duplex DNA is added to the
reaction, the k
drops abruptly to about 20
min
, and remains at that level throughout the
ensuing DNA strand exchange reaction as long as ATP is regenerated
(Schutte and Cox, 1987; Ullsperger and Cox, 1995). The reaction is
apparently quite inefficient, with about 100 ATPs hydrolyzed per base
pair of hybrid DNA created. The function of this ATP hydrolysis is
incompletely understood. An important clue was provided by the
observation that RecA protein can promote DNA strand exchange under
some conditions in the presence of ATP
S, an ATP analog that is not
readily hydrolyzed by RecA (Menetski et al., 1990; Rosselli
and Stasiak, 1990). This observation was reinforced more recently by
work on the RecA mutant K72R (Rehrauer and Kowalczykowski, 1993) and
again with wild type RecA protein in the presence of
ADP-AlF
(Kowalczykowski and Krupp, 1995).
The Lys
Arg substitution in the K72R mutant occurs in a well
conserved nucleotide binding fold, and the mutant protein binds but
does not hydrolyze ATP. The mutant will also promote a limited DNA
strand exchange, functioning best with dATP (Rehrauer and
Kowalczykowski, 1993). These results demonstrate that the RecA filament
has an inherent capacity to take up at least three DNA strands and
promote DNA strand exchange without ATP hydrolysis, and have been used
to argue against an essential role for ATP hydrolysis in DNA strand
exchange. The RecA filament tends to stabilize the hybrid DNA products
of a DNA strand exchange reaction (Adzuma, 1992).
Why, then, does
RecA protein hydrolyze ATP? One way to elucidate the function of
RecA-mediated ATP hydrolysis is to define the limitations of reactions
that occur without it. A series of studies determined that the strand
exchange occurring with ATPS is limited in extent and
bidirectional (Jain et al., 1994; Konforti and Davis, 1992).
The ATP
S reactions also did not proceed past structural barriers
in the DNA (Kim et al., 1992a; Rosselli and Stasiak, 1991) and
did not accommodate four DNA strands (Kim et al., 1992b). ATP
hydrolysis therefore appears to confer several important properties to
the DNA strand exchange reaction, being required in particular for
unidirectional branch movement that can bypass barriers and for
four-strand exchange reactions. These observations provide some
indirect mechanistic clues but have been obtained only under the
conditions used with ATP
S.
The properties of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis provoke questions which, if answered, might shed additional light on ATP function and strand exchange mechanism. In studies to date with three-strand exchange reactions using homologous substrates, there is a rapid formation of a limited segment of hybrid DNA product (typically 1-3 kbp). However, at this point the reaction halts or slows dramatically. Since the entire filament is capable of promoting an exchange between homologous substrates, it is surprising that the entire reaction does not proceed to completion. The slowing or cessation of strand exchange implies that discontinuities exist in some component of an early strand exchange intermediate. The one possibility presented to date involves filament discontinuities, with ATP hydrolysis needed to recycle RecA monomers and correct the discontinuities (Menetski et al., 1990; Rehrauer and Kowalczykowski, 1993; Kowalczykowski and Krupp, 1995). As shown below, the only reasonable alternative involves discontinuities in a key DNA structure that serves as a strand exchange intermediate.
A similar
and related unresolved question can be defined even under conditions in
which ATP is being hydrolyzed. Upon addition of a homologous duplex DNA
to RecAssDNA complexes hydrolyzing ATP, the rate of ATP
hydrolysis declines abruptly by up to 30%. The observed decline is
directly proportional to the length of homologous sequence in the
duplex, providing evidence that the entire length of available homology
is detected within a minute or two with direct DNA-DNA interactions
occurring over distances of 8 kbp or more (Schutte and Cox, 1987).
However, productive strand exchange detectable after RecA removal from
the DNA proceeds much slower, requiring 20 min or more to encompass the
same 8 kbp. There again appears to be a fast phase of strand exchange
in which some short length of hybrid DNA is generated, followed by a
slow phase in which the nascent hybrid DNA is extended. The fast phase
is sometimes manifested as an apparent burst phase in hybrid DNA
formation when ATP is hydrolyzed (Kahn and Radding, 1984; Bedale and
Cox, 1996). As in the cases where ATP is not hydrolyzed, it is
necessary to explain why the fast phase comes to an end before strand
exchange is complete, even though the response of the filament
indicates the detection of homology along the entire length of the DNA.
In this report, we further explore the properties of the fast and
slow reaction phases and present a simple model that explains why the
fast phase is limited in extent. The model also explains all properties
of the two reaction phases and applies to reactions carried out with or
without hydrolysis of ATP. The results complement and/or confirm a
number of previous observations obtained with ATPS, using RecA
K72R employed under more classical reaction conditions, and further
characterize the RecA K72R mutant protein. To date, many aspects of the
DNA strand exchange reaction promoted by the RecA K72R mutant protein
remain unexplored, but have the potential to test many of the ideas
outlined above about the role of the RecA ATPase activity.
After the gel was stained with ethidium bromide (1 µg/ml) for at least 30 min and destained for at least 2 h, the gel was then photographed over an ultraviolet transilluminator. The intensities of DNA bands were quantified by scanning the photographic negatives using a Molecular Dynamics Personal Densitometer SI and analyzing the image with ImageQuant software (Version 4.2). In order to correct for variability in sample loading onto the agarose gel, the band corresponding to full-length products and/or the broad smear representing intermediates of the strand exchange reaction were quantified as the fraction of the total fluorescing DNA in a given gel lane.
In some experiments, the
data was plotted with respect to the concentration of Mg in excess of that involved in a complex with dATP. The
concentration of ``excess'' Mg
was
calculated based on the reported dissociation constant of 1
10
M for the Mg
ATP complex (Alberty,
1969).
Three types of information were derived from the electron microscopy experiments. First we wished to confirm that the reaction intermediates observed on gels had the anticipated structure. Representative molecules are shown for some experiments and results described in the text. Second, we wished to determine the proportion of the duplex linear substrates that was involved in DNA strand exchange reactions leading to intermediates. This was done by counting the intermediates and unreacted linear duplex DNA molecules found in a representative sample from each experiment. Complex recombinational events involving more than two DNA substrate molecules and events that were interpreted to arise from broken or nicked substrates (the latter produce low levels of complex reaction products in the reactions) were ignored in these estimates. In some experiments, the grids from different reactions were assigned an undescriptive identification code by Q. S., and were subsequently counted in a random sequence by R. B. I. Third, it was important to estimate the length of hybrid DNA generated for a representative sample of intermediates in some experiments. Because of the large numbers of samples, obtaining accurate measurements of significant numbers of intermediates in all of them was impractical; we have therefore estimated the degree of exchange as described earlier (Jain et al., 1994). Briefly, the ratio of unexchanged to exchanged duplex DNA was judged. These ratios were then converted into base pairs of exchanged DNA using the known length of the linear dsDNA substrate. The data were sorted into 8-10 degrees of exchange and plotted as histograms. Degrees of exchange with the 1.3-kbp DNA fragment used as a substrate in some experiments was divided in this manner into eight equal segments of 165 bp; linear M13mp8.1037 dsDNA was divided into 10 segments of 830 bp.
These judgments were checked in two ways. First,
two grids each from two different samples were counted and judged
``blind'' as described above. The four sets of data were then
compared by the two-way contingency test at the 95%
confidence level. In both cases, the data sets for the two samples were
not significantly different. The judgments have also been checked
directly by comparing the data obtained to data from more detailed
measurements done on the same samples (Jain et al., 1994).
Statistical analysis again showed that there is no significant
difference in the result obtained with the two methods.
Copies of the electron micrographs discussed in this publication are available for viewing or downloading using a World-Wide Web client at the URL: http://phage.bocklabs.wisc.edu/.
Figure 2:
DNA strand exchange reactions promoted by
RecA K72R. Reactions were carried out as described under
``Materials and Methods,'' with 6.7 µM wtRecA or
RecA K72R proteins, 2 µM SSB, 20 µM X174 circular ssDNA, and 20 µM linear
X174 dsDNA (cleaved with PstI). Reactions also contained
3 mM ATP (A) or dATP (B). In Panel
A, lane M
contains
X174 linear
duplex DNA as a marker, and M
contains
supercoiled and nicked circular
X174 DNA. In Panel B,
the markers are
X174 ssDNA (M
) and
linear duplex DNA (M
). In the reactions
themselves, time points are 0, 10, 20, 40, 60, 90, 120, 150, and 180
min, respectively, left to right. Labels are: P, the nicked
circular duplex product of DNA strand exchange; S, the linear
duplex substrate; ss, the circular ssDNA substrate; and I, reaction intermediates.
Figure 3:
RecA K72R-mediated DNA strand exchange is
affected by the concentration of magnesium ion. Reactions (20 µl)
were carried out for 3 h as described under ``Materials and
Methods'' and contained 6.7 µM RecA K72R protein, 2
µM SSB, 20 µM M13mp8 circular ssDNA, and 20
µM linear M13mp8 dsDNA (cleaved with SmaI). The
intermediates and products were quantified as described under
``Materials and Methods.'' Panels A and B show reactions with dATP concentrations at 3 and 12 mM,
respectively, and Mg concentrations indicated at the
top of the gels. Labels are as described in Fig. 2. Panels C and D show the quantified results of several experiments
plotted as a function of total Mg
concentration, with
full-length hybrid DNA products shown in C and intermediates
plotted in D. In Panels E and F, the same
results are plotted as a function of excess Mg
concentration (relative to that involved in a Mg
dATP
complex), calculated as described under ``Materials and
Methods.'' The dATP concentrations in Panels C-F are: (
), 1 mM; (
), 3 mM; (
), 6
mM; (
), 9 mM; (
) 12 mM. The
marker lane (M) contains supercoiled and nicked circular
M13mp8 DNA, providing a marker for the full-length nicked circular
products of DNA strand exchange.
The results indicate that the K72R mutant protein can promote a
complete strand exchange reaction generating over 7 kbp of hybrid DNA
at significant levels. Excess Mg beyond that in the
Mg
dATP complex is required for optimal uptake of substrate duplex
DNA into the nucleoprotein filament formed on ssDNA to form strand
exchange intermediates, but the same excess Mg
inhibits the formation of the extensive lengths of hybrid DNA
needed to generate the completed products. The weak uptake of substrate
duplex DNA when excess Mg
concentrations are low
appears to be one factor limiting the yield of full-length products
under conditions otherwise optimal for their generation.
As shown in Fig. 4, the generation of full-length strand exchange products
by the K72R mutant is unsynchronized (in different nucleoprotein
filaments) and very slow. Products appear slowly over an 8-h time
course. Under optimal Mg conditions (where the
concentrations of dATP and Mg
are approximately
equal), 10-20% of the input duplex DNA was readily converted to
full-length products in 8 h. The yield of products is still increasing
at 8 h and may be limited only by time and the stability of the
nucleoprotein filaments. When significant levels of free Mg
were present, the generation of full-length products was greatly
reduced even over a long time course. In the presence of 3 mM dATP, products were generated at 4 mM Mg
, but not at 6 mM or above with
substrates derived from M13mp8. The generation of intermediates peaked
at 6 mM Mg
, and appeared to decrease
somewhat at higher Mg
concentrations, although the
yield was still considerable at 10 mM.
Figure 4:
Generation of full-length hybrid DNA
products by RecA K72R is slow. Reactions (100 µl) were carried out
as described in Methods, with 6.7 µM RecA K72R proteins, 2
µM SSB, 20 µM M13mp8 circular ssDNA, and 20
µM linear M13mp8 dsDNA (cleaved with SmaI).
Reactions also contained 3 mM dATP. Panels A and B show the reactions with 4 and 10 mM Mg, respectively. Time points are 0, 1, 2, 3, 4,
5, 6, and 8 h, respectively, left to right. Labels are as described in Fig. 2. The marker lanes (M) contain supercoiled and
nicked circular M13mp8 DNA as in Fig. 3. Panel C is a
plot of the quantified full-length product formation for the reactions
in Panels A and B. Panel D shows the quantified
formation of joint molecule intermediates in an expanded set of
reactions including those in Panels A and B. Symbols
for Mg
concentrations in Panels C and D are: (
), 2 mM; (
), 4 mM; (
),
6 mM; (
), 8 mM; (
), 10
mM.
The limits to the
length of hybrid DNA that can be formed by the RecA K72R mutant protein
in the presence of 10 mM magnesium acetate was explored
further (data not shown). The RecA K72R mediated-reaction generated
completely exchanged products with a 1.3-kbp duplex substrate. A
similar reaction with a 2.9 kbp DNA fragment derived from M13mp8 (the
small fragment from ClaI digestion) exhibited some product
formation even in the 10 min time point, but the reaction was much
weaker than that promoted by the wtRecA protein. When the 4.3-kbp ClaI fragment of M13mp8 was used as the duplex substrate, no
product generation was detected (data not shown). As noted in Fig. 2, limited product formation was sometimes promoted by RecA
K72R with somewhat longer DNAs when the substrates were derived from
X174. We concluded that the mutant protein could promote the
rapid generation of over 1 kbp of hybrid DNA, with extended hybrid DNA
regions observed at efficiencies declining rapidly as a function of
length, when the dATP and Mg
concentrations were 3
and 10 mM, respectively.
Binding of SSB to ssDNA prior to addition of the K72R mutant protein eliminated the production of strand exchange products under all conditions, and also inhibited the formation of strand exchange intermediates (data not shown). Optimal reactions with the mutant protein are observed only when it is added prior to the SSB. The generation of full-length strand exchange products under optimal conditions also required a stoichiometric concentration of the RecA K72R mutant protein relative to the ssDNA. The generation of full-length products did not increase at all when the concentration of mutant protein exceeded the stoichiometric level of one monomer per three nucleotides of ssDNA (data not shown). Excess protein, which might fill or eliminate discontinuities in the filaments, does not improve the reaction.
To further test the idea
that free Mg stimulates formation of intermediates
but inhibits subsequent branch migration needed to generate products,
experiments were carried out in which strand exchange was initiated at
one Mg
concentration, and then shifted by the
addition of more Mg
or dilution. The dATP
concentration was set at 3 mM. As shown in Fig. 5,
addition of Mg
to reactions initiated under
conditions optimal for product formation (3 mM Mg
) resulted in strong inhibition. In this case,
the added free Mg
would be expected to block the
branch migration needed to generate products. Contrasting to a degree,
the complementary dilution experiment did not always produce the
anticipated restoration of product formation. When the reaction was
initiated at 6 mM Mg
, then diluted 1 h later
(after intermediates had formed) so as to make the Mg
concentration equal to that of the dATP, product formation was
stimulated only slightly. The reaction shown after the dilution in Fig. 5B was not nearly as strong as that observed in
reactions initiated with 3 mM Mg
, indicating
that the use of excess Mg
produces a degree of
hysteresis. The same result was observed when EDTA was used to remove
excess Mg
(data not shown). The hysteretic effect of
excess Mg
was time-dependent, since a dilution at
only 5 min after the reaction was initiated fully restored product
formation comparable to that observed in reactions without excess
Mg
(data not shown). The results suggest that when
the concentration of Mg
exceeds that of dATP,
intermediates are formed that slowly take on a structure that is not
easily resolved when Mg
concentrations are lowered.
Figure 5:
Generation of full-length hybrid DNA
products can be stimulated or blocked by adjusting magnesium ion
concentration. Reactions were carried out as described under
``Materials and Methods.'' Reactions contained 6.7 µM RecA K72R protein, 2 µM SSB, 20 µM M13mp8 circular ssDNA, and 20 µM linear M13mp8 dsDNA
(cleaved with SmaI). Reactions also contained 3 mM dATP. In Panel A, two reactions (100 µl) are shown
containing 3 mM magnesium acetate. One hour after the
reactions were initiated, 2 µl of 10 mM Tris acetate (80%
cation, pH 7.5) or concentrated magnesium acetate was added to the
reactions at left and right, respectively, bringing the final
Mg concentration in the reaction on the right to 8
mM. The reaction time points are 0, 1, 2, 3, 4, 5, 6, and 8 h,
respectively, left to right, and the additions were made immediately
after the 1-h time point shown. In Panel B, a single reaction
(120 µl) was started in the standard reaction buffer containing 6
mM magnesium acetate. After taking the 0- and 1-h time points (lanes 1 and 2), the reaction was divided into two
40-µl aliquots. Each aliquot was diluted 1:1 into a buffer
containing 25 mM Tris acetate (80% cation, pH 7.5), 3 mM potassium glutamate, 1 mM DTT, 5% (w/v) glycerol, 3
mM dATP, and a dATP regeneration system (11.8 mM phosphoenolpyruvate, 20 units ml
pyruvate
kinase), and either 6 mM magnesium acetate (reaction
6) or no magnesium acetate (reaction
3). RecA
K72R, SSB, and DNA substrates in these reactions were diluted 2-fold.
The reactions proceeded for additional 7 h, with the five gel lanes in
each set representing 2-, 3-, 4-, 6-, and 8-h time points,
respectively, left to right. Labels are as described in Fig. 2.
The marker lane contains supercoiled and nicked circular M13mp8 DNA as
in Fig. 3.
With wtRecA protein, the duplex DNA with
proximal homology was converted efficiently into a slowly migrating
product, previously identified as a branched molecule in which strand
exchange has proceeded to the homology/heterology junction, creating
7.2 kbp of hybrid DNA (Jain et al., 1994). When homology is
restricted to the distal end, the reaction is weaker (Fig. 6A), and lengths of hybrid DNA produced are much
shorter (Jain et al., 1994). RecA K72R protein-mediated strand
exchange produced intermediates whether the homology was located on the
proximal or distal ends, with little evident bias (Fig. 6A). There was no significant change in these
results when the reactions were cross-linked with AMT prior to
electrophoresis to eliminate spontaneous branch migration, or when the
Mg concentration was lowered to 3 or 6 mM,
although the yield of intermediates declined with the lower
Mg
concentrations (data not shown).
Figure 6: DNA strand exchange is bidirectional with RecA K72R. Reactions were carried out as described under ``Materials and Methods,'' with 6.7 µM wtRecA or RecA K72R proteins, 2 µM SSB, 20 µM M13mp8 circular ssDNA, and 20 µM linear M13mp8.1037 dsDNA. The M13mp8.1037 DNA was cleaved with either EcoRI (distal homology) or BamHI (proximal homology). Reactions also contained 3 mM dATP. For each reaction, time points are 0, 10, 30, 60, and 90 min, respectively. Labels are as in Fig. 2, with ss designating the circular ssDNA substrate. Panels B and C are the RecA K72R-mediated reactions with distal and proximal homology, respectively. An aliquot from a 60-min reaction mixture was removed, cross-linked, deproteinized, and analyzed by electron microscopy as described under ``Materials and Methods.'' Lengths of DNA exchanged in intermediates produced by RecA K72R, using a judgment procedure described under ``Materials and Methods.''
The branched DNA intermediates formed in these reactions were examined by electron microscopy, and the approximate lengths of hybrid DNA in each molecule determined. The results (Fig. 6, B and C) confirm that the reaction with RecA K72R proceeded with no substantial bias on either end of the duplex substrate, and produced only limited lengths of hybrid DNA. We conclude that strand exchange mediated by the K72R mutant is bidirectional.
Figure 7:
RecA K72R-mediated DNA strand exchange
does not bypass structural barriers in the duplex DNA substrate.
Agarose gel assays were carried out as described under ``Materials
and Methods.'' Reactions contained 6.7 µM wtRecA or
RecA K72R proteins, 2 µM SSB, 3 mM dATP, 20
µM M13mp8 (left) or M13mp8.52 (right)
circular ssDNA, and 10 µM of the 1323-bp linear duplex
substrate derived from M13mp8.52. In the reaction illustrated on the
right, both substrates contain the 52-bp insert, and thus they are
homologous throughout their length. DNA markers in lane M are
DNA digested with BstEII. The reaction time points for
all four reactions are 0, 10, 20, 40, 60, 90, and 180 min, from left to
right. The labels are: I, reaction intermediates; P,
gapped circular DNA molecules produced by a complete strand exchange; ss, circular ssDNA substrate; S, 1323-bp linear
duplex fragment used as a substrate.
In the RecA K72R reactions (Fig. 7), the reaction of the 1.3-kbp duplex DNA fragment with
circular M13mp8 ssDNA produced reaction intermediates that accumulated
with time, but no complete products. In contrast, significant product
formation was observed for the completely homologous reaction using
M13mp8.52 ssDNA. Therefore, a 52-bp heterologous insert in the duplex
DNA blocked RecA K72R-mediated DNA strand exchange. Even in the
completely homologous reaction, the generation of completely exchanged
products was weak with the K72R mutant, and reaction intermediates were
still the predominant species at the end of the reaction. Both of the
reactions proceeded much better in the presence of wtRecA protein and
dATP. Substantial amounts of the completely exchanged product were
produced even when the duplex contained the heterologous insertion. The
Mg concentration had no effect on the capacity of the
mutant protein to bypass the barrier (data not shown). In a series of
reactions carried out with Mg
concentrations ranging
from 2 to 10 mM, intermediates were produced in significant
quantities but no completed products were seen with the mutant protein
under any conditions.
These reactions were examined by electron microscopy at the 40-min time point (Fig. 8). In the RecA K72R-mediated reaction, 121 intermediates but no completed products were found in 362 randomly chosen duplex or partial duplex molecules. Several different types of intermediates were found (Fig. 8, A-D), with 101 (84%) identified as the standard type (Fig. 8, A and B), and 20 (16%) falling into a more complex class in which strand exchange appeared to have progressed from both ends without unwinding the 52-bp insert (Fig. 8D). The molecules in the latter class were observed at similar levels in every repetition of this experiment. Their probable origin is described under ``Discussion'' in the context of a broader model for DNA pairing. In the wtRecA-mediated reaction, 29 (9%) of 327 randomly chosen duplex or partial duplex molecules were in the product form (Fig. 8C), 91 (28%) were standard intermediates, and 7 (2%) were intermediates with more complex structures.
Figure 8: Electron microscopy of DNA species found in reactions involving a structural barrier in the duplex substrate. Samples taken 40 min into reactions such as those in Fig. 10(carried out under identical reaction conditions) were cross-linked with AMT, deproteinized, and spread as described under ``Materials and Methods.'' Panels A and B show typical reaction intermediates found in the reaction with RecA K72R. At the right of these panels are shown examples of the circular ssDNA and linear dsDNA substrates, respectively. Panel C shows a reaction product found in the reaction with wtRecA. Panel D shows a class of reaction intermediate described in the text, in which strand exchange appears to have progressed from both ends of the linear duplex substrate. Such molecules were observed in both the wtRecA and RecA K72R reactions (this one is from the RecA K72R sample). Panel E gives lengths of DNA exchanged in intermediates produced by RecA K72R, using a judgment procedure described under ``Materials and Methods.''
Figure 10: A model explaining the cessation of the rapid phase of strand exchange in the context of a discontinuous DNA pairing intermediate. Formation of the hypothetical DNA pairing intermediate shown in Panel V is illustrated in five steps. I, pairing is initiated at one end of a duplex DNA substrate. Extension of the paired region requires the rotation of both the filament and the duplex DNA, as shown by circular arrows. II, as the paired region lengthens, some probability exists for an intramolecular pairing interaction elsewhere in the filament (black arrow). III, pairing at the new location creates a new point for continued spooling of the duplex DNA into the filament. However, a segment of DNA is left outside of the filament as an external loop by the second pairing initiation. IV, multiple loops can form (e.g. segments B-C and D-E), with paired segments (e.g. C-D) between them. V, resolution of the loops requires their rotation about the axis of the RecA nucleoprotein filament.
The extent of strand exchange was also quantified for the reaction by RecA K72R (Fig. 8E). Of 101 randomly chosen standard intermediates (Fig. 8, A and B), 52% had halted in the middle of the linear duplex DNA, and the remainder had shorter regions of hybrid DNA. One molecule was found in which strand exchange appeared to have bypassed the insert (we attribute an incidence this low to an artifact produced by the low level of nicked or broken DNA molecules present in every DNA preparation). We conclude that RecA K72R protein will not promote bypass of heterologous insertions during DNA strand exchange.
Figure 9:
Four-strand exchange reactions are not
promoted by RecA K72R. Reactions were carried out as described under
``Materials and Methods,'' and contained 3 µM wtRecA or RecA K72R proteins, 0.6 µM SSB, 3 mM dATP, 12 µM gapped duplex DNA substrate (GD1037), and 10 µM of the 7834-bp linear duplex
substrate, generated by NcoI and EcoRI cleavage of
M13mp8.1037 (8226 bp). The linear duplex overlaps the single strand gap
in the gapped duplex by 605 bp. Panel A shows the reactions
monitored with a agarose gel. Markers (M) are bacteriophage
DNA digested by BstEII. The time points for both
reactions are 0, 10, 30, 60, and 90 min, respectively, left to right.
Labels are: GD1037, the gapped duplex substrate; S,
the 7834-bp linear duplex substrate; P, the GD432 gapped
duplex product generated by a complete strand exchange reaction and
linear duplex DNA with 7229-bp duplex region and a 605-base
single-stranded tail; I, reaction intermediates. Panels
B-D, samples taken at 60 min into the reaction were
cross-linked with AMT, deproteinized, spread, and examined by electron
microscopy. The labels a and b are explained in the
legend for Panel E. B and C, typical reaction
intermediates generated in the reaction with RecA K72R. D, a
Holliday intermediate generated in the reaction with wtRecA protein.
The Holliday junction, slightly denatured to display the individual
strands, is labeled HJ. Panel E gives a schematic focusing on
two stages of the strand exchange reaction. First, the reaction is
initiated as a three-strand reaction in the single strand gap,
producing a branched molecule with a short displaced single strand
labeled a. In the substrates used, the linear duplex overlaps
the gap by 605 bp, leaving a 432-bp region of ssDNA that is not
included in the region undergoing exchange (labeled b). In the
wtRecA-mediated reaction, the branch moves into the neighboring duplex
region of the gapped duplex, producing a Holliday intermediate as
shown, and ultimately a complete strand exchange. The a and b labels remain the same.
Our primary conclusions are: (a) that there are two
phases to a RecA-mediated DNA strand exchange without ATP hydrolysis,
with different Mg requirements, and (b) that
the RecA protein-mediated DNA strand exchange is severely constrained
when ATP is not hydrolyzed. The RecA K72R protein, which does not
hydrolyze dATP or ATP at detectable levels, will promote the generation
of DNA products with over 7 kbp of hybrid DNA. However, the reaction is
slow and greatly affected by the concentration of Mg
.
Under all conditions the reaction is also bidirectional, will not
bypass heterologous insertions in the duplex substrate, and will not
accommodate four DNA strands. This last set of limitations are seen
with wild type RecA protein in the presence of ATP
S (Rosselli and
Stasiak, 1991; Kim et al., 1992a, 1992b; Konforti and Davis,
1992; Jain et al., 1994). Many of the results with the mutant
protein were obtained under conditions typical of reactions with wild
type protein and ATP.
A mechanistic context for further discussion of the results is provided by the model in Fig. 10. The model is designed to explain the observed limitations to the lengths of hybrid DNA generated during RecA-mediated DNA strand exchange when ATP is not hydrolyzed. As an alternative to the discontinuous RecA filaments proposed by Kowalczykowski and colleagues (Menetski et al., 1990; Rehrauer and Kowalczykowski, 1993; Kowalczykowski and Krupp, 1995), we suggest that the discontinuity is instead found in a key DNA pairing intermediate. In any DNA strand exchange reaction with RecA protein (with or without ATP hydrolysis), initiation is presumed to occur via the alignment of a ssDNA within the filament with a homologous duplex to form a pairing intermediate with all three strands interwound (structure unspecified for purposes of this discussion). This must involve a spooling of the duplex into the filament groove, with both the filament and DNA rotating in solution as shown in Fig. 10. If the rotation and accompanying spooling proceed uninterrupted, a uniform DNA pairing intermediate would be created throughout the length of the DNA substrates. Since the filament stabilizes the hybrid DNA products of strand exchange, this intermediate would be rapidly converted to hybrid DNA throughout its length. However, the entire RecA filament is set up to initiate DNA pairing. Once pairing is initiated at one location along the filament, a pairing interaction at another location in the same filament becomes intramolecular and much more likely. As spooling lengthens the initial pairing interaction to some point B (Fig. 10), pairing at another point C will initiate another segment of DNA pairing intermediate that can be lengthened by spooling as was the first. Further spooling at point B will then be blocked, because the duplex DNA between points B and C has been constrained at point C by the new pairing interaction. We define the segment between points B and C as an external loop. The pairing process could generate any number of such loops along the length of a paired duplex DNA. The loops may be long or very short, and the average distance between them would reflect the efficiency of intramolecular DNA pairing under a given set of reaction conditions. We propose that formation of a pairing intermediate with alternating loops and paired regions along the entire length of available homology defines the rapid phase of DNA strand exchange under conditions generally used for RecA reactions. Because of topological constraints, the only stable and productive strand exchange in such an intermediate (where one strand of the duplex substrate can be displaced) would occur between an end of the duplex and the beginning of the first loop, such as the A to B segment in Fig. 10. If the reaction shown was terminated at Panel V, the A-B segment contains the only stable hybrid DNA that would remain after protein removal.
In this model, the A-B segment defines the extent of hybrid DNA formation in the rapid phase of strand exchange. Extension of the A-B segment requires the rotation of the loop around the filament axis, with the loop DNA axis more or less parallel to the filament axis, so that DNA is wound into the filament groove at one end of the loop and out of the filament at the other end. Since a given paired region is lengthened only at the expense of another paired region (e.g. the A-B segment can lengthen at the expense of the C-D segment), this process is inevitably much slower than the rotary diffusion/spooling process that generates the various paired regions in the first place. In the bottom panel of Fig. 10, V, if the viewer looks down the filament axis from the left side, and rotates the loop clockwise about the axis as shown, the loop will migrate away from the viewer (or to the right as it is drawn). Counterclockwise rotation will move the loop in the opposite direction. The rate of any migration that occurred would be limited or blocked altogether by steric interference, the stability of neighboring paired segments, and other factors. In vitro, some of the ``loops'' would inevitably be intermolecular, spanning different filaments and creating the aggregate networks first described by Radding and colleagues (Tsang et al., 1985).
This
scenario is consistent with the observed effects of Mg on the reaction promoted by RecA K72R. Whereas increased
Mg
concentrations have a destabilizing effect on
protein-DNA interactions, they can have a stabilizing effect on the
pairing interactions between DNA strands (Record and Spolar, 1990;
Record, 1975), including triplex DNA structures (Kohwi and Kohwi, 1988;
Wells et al., 1988; Malkov et al., 1992; Shchyolkina et al., 1994). Concentrations of Mg
in
excess of that required to form Mg
dATP complex should therefore
facilitate the initial formation of pairing intermediate in the rapid
phase (and the formation of additional paired regions to generate
loops), leading to an enhancement of joint molecule formation. However,
since extension of the stable hybrid DNA in the joint molecule must
come at the expense of other paired segments, stabilization of the
other paired segments by the excess Mg
will tend to
block extension and the formation of completely exchanged products. The
formation of complex structures with multiple external loops might
block resolution of the intermediates to products even when the
Mg
concentration was subsequently reduced, leading to
the observed hysteresis in reactions initiated with excess
Mg
and then diluted (Fig. 5). When the
Mg
concentration is just sufficient to form the
Mg
dATP complex, the decreased pairing efficiency could reduce the
number of external loops and allow better production of completed
strand exchange products over time. All of these effects are observed.
The effects of Mg
concentration on loop migration are
analogous in many respects to the effects of Mg
on
spontaneous DNA branch migration in solution. DNA branch movement
requires the formation of base pairs on one side of the branch at the
expense of base pairs on the other side, and the rate of this process
is reduced by up to 3 orders of magnitude by added Mg
(Panyutin and Hsieh, 1994).
We also routinely observe molecules by electron microscopy that must be formed by a process like that illustrated in Fig. 10. If the homologous duplex DNA substrate is sufficiently short, a limiting case might be observed where pairing was initiated at one end, and then a single external loop was sometimes formed followed by extension of the three-stranded DNA pairing intermediate out to the opposite end of the duplex. Stable strand displacement could then be seen after protein removal that appears to proceed from both ends, held together by an unexchanged loop of substrate duplex as in the molecule shown in Fig. 8D. Note that this type of molecule cannot form by independent pairing initiation at the two ends, since simultaneous pairing at either end and extension of both paired segments toward the center is topologically forbidden (the duplex DNA would have to rotate in opposite directions to extend each paired region). If one paired segment is initiated at the left end of the duplex and extended to the right, the second paired segment must be initiated away from the right end and extended to the end from left to right.
When ATP is hydrolyzed, the nascent hybrid duplex DNA is extended unidirectionally. The external loops would have to be rotated uniquely in one direction to bring this about. Elsewhere, we have proposed a model for how ATP hydrolysis might be coupled to such a rotation of external DNA relative to the filament axis (Cox, 1994). ATP hydrolysis also permits the bypass of barriers. A four-strand exchange reaction will not occur at all unless ATP is hydrolyzed. These properties can best be rationalized in the context of RecA's function in recombinational DNA repair (Clark and Sandler, 1994; Cox, 1993).
We note that even if filament discontinuities occur and help to limit DNA pairing in the absence of ATP hydrolysis, the DNA loops we describe above can still be formed when duplex DNA is paired at two separated filament segments (and may be inevitable). These loops would have to be resolved irrespective of any redistribution of RecA protein monomers, and their resolution may require ATP hydrolysis.
The
observed effects of Mg suggest that the reactions
with the K72R mutant are not seriously limited by filament
discontinuities. Full-length hybrid DNA products are generated, albeit
slowly, at appropriate Mg
concentrations. Excess
mutant protein, which might plug any gaps in a discontinuous filament,
has no effect on the reaction.
The absolute requirement for ATP hydrolysis in the four-strand exchange reaction is also potentially instructive in discriminating between mechanistic alternatives for the slower hybrid DNA extension phase. The segments of RecA filament present when ATP is not hydrolyzed in a hypothetical discontinuous filament cannot promote a four-strand exchange under any conditions, and a simple redistribution of RecA monomers to create a contiguous but otherwise identical filament at other locations should not change this result. Many lines of evidence indicate that the RecA filament can only assimilate three DNA strands (Cox, 1993, 1995). A four-strand exchange reaction therefore requires a contribution from ATP hydrolysis that goes beyond the turnover of RecA filament complexes already bound to hybrid DNA product. The proposal that RecA-mediated ATP hydrolysis is coupled to a coordinated rotation of DNA molecules to bring about branch movement during strand exchange provides a mechanism to explain the promotion of four-strand exchanges by a RecA filament that can only assimilate three DNA strands (Kim et al., 1992b; Cox, 1994).
The RecA K72R mutant protein is surprisingly competent in the
promotion of DNA strand exchange reactions in vitro. It
fulfills the requirements of a DNA pairing activity, which in some
scenarios would generate branched recombination intermediates before
yielding to specialized branch migration activities such as RuvAB or
RecG (West, 1992; Kowalczykowski et al., 1994). However, cells
in which the wild type recA gene is replaced by a recA K72R gene
display a recA phenotype. They are as deficient in
homologous recombination, as sensitive to UV radiation, and as unable
to induce the SOS response as a recA null mutant (Konola et
al., 1994). (
)These results indicate that the ATPase
activity of RecA is important in vivo. The point in
recombinational processes where RecA is replaced by RuvAB or RecG is
currently undefined.
The RecA K72R mutant protein has the capacity
to facilitate the cleavage of LexA repressor in vitro,
especially in the presence of ATPS. The defect that the K72R
mutation confers on cells in SOS induction can be explained by the
slower kinetics of LexA autocatalytic cleavage with the K72R mutant in
the presence of ATP and/or dATP.