From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
Received for publication, December 18, 2002, and in revised form, February 10, 2003
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ABSTRACT |
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Optimal conditions for RecA protein-mediated DNA
strand exchange include 6-8 mM Mg2+ in
excess of that required to form complexes with ATP. We provide evidence
that the free magnesium ion is required to mediate a conformational
change in the RecA protein C terminus that activates RecA-mediated DNA
strand exchange. In particular, a "closed" (low Mg2+)
conformation of a RecA nucleoprotein filament restricts DNA pairing by
incoming duplex DNA, although single-stranded overhangs at the ends of
a duplex allow limited DNA pairing to occur. The addition of excess
Mg2+ results in an "open" conformation, which can
promote efficient DNA pairing and strand exchange regardless of DNA end
structure. The removal of 17 amino acid residues at the
Escherichia coli RecA C terminus eliminates a measurable
requirement for excess Mg2+ and permits efficient DNA
pairing and exchange similar to that seen with the wild-type protein at
high Mg2+ levels. Thus, the RecA C terminus imposes the
need for the high magnesium ion concentrations requisite in RecA
reactions in vitro. We propose that the C terminus acts as
a regulatory switch, modulating the access of double-stranded DNA to
the presynaptic filament and thereby inhibiting homologous DNA pairing
and strand exchange at low magnesium ion concentrations.
The RecA protein of Escherichia coli plays a central
role in the processes of homologous DNA recombination and DNA repair. RecA is a DNA-dependent ATPase that catalyzes an in
vitro DNA strand exchange reaction between single-stranded
(ssDNA)1 and homologous
double-stranded DNA (dsDNA) molecules. The DNA strand exchange reaction
takes place in several stages (Fig. 1). The RecA protein forms a
nucleoprotein filament that completely encompasses the circular ssDNA.
This filament then aligns the bound single strand with a homologous
duplex DNA to form a DNA pairing intermediate often referred to as a
joint molecule. 1000 base pairs of DNA can be aligned and exchanged in
a joint molecule under the empirically defined optimal reaction
conditions, which typically include 1-3 mM ATP and about
10 mM magnesium ion. All steps to this point, including the
formation of joint molecules, require ATP but not ATP hydrolysis. ATP
hydrolysis is needed only to complete the late stages of strand
exchange of long DNA substrates, often derived from bacteriophage DNAs.
Whereas DNA pairing, leading to joint molecule formation, can occur at
either end of a linear duplex, the subsequent and ATP
hydrolysis-dependent extension of the nascently paired
regions is unidirectional, proceeding 5' to 3' relative to ssDNA
initially bound in the filament. Thus, exchange proceeds in one
direction along the linear duplex, and joints formed at the "wrong"
end in the pairing phase are eliminated. In a DNA strand exchange
involving quite long DNAs that leads to nicked circular product
formation, the ends of the duplex where the exchange begins and ends
are referred to as proximal and distal, respectively (Fig.
1) (1, 2).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DNA strand exchange promoted by the RecA
protein in vitro. The DNA strand exchange
reaction used in this report is illustrated. DNA strand exchange occurs
in multiple stages. A RecA filament first forms on the single-stranded
DNA (not shown). A linear duplex is then aligned with the bound single
strand, and a strand exchange is initiated. The reaction can occur on
either end of the duplex, and intermediates formed at either end can be
observed when an ATP analogue that is not hydrolyzed (ATP S) is used.
The subsequent extension of the hybrid DNA proceeds unidirectionally
when ATP is hydrolyzed, 5' to 3' relative the single-stranded circle.
Thus, intermediates formed on one end (proximal) proceed to products,
whereas those formed on the other (distal) end are eliminated when ATP
is hydrolyzed. The branched reaction intermediates are referred to as
joint molecules. The product used most often to analyze reaction
progress is the nicked circular duplex. The S1,
S2, I, and P labels identify DNA species that are similarly
labeled in the data figures.
Examination of the conditions for an optimal RecA protein-catalyzed DNA
strand exchange reaction reveals an unexplored enigma. Magnesium forms
a relatively strong 1:1 complex with ATP (3) and should be required at
concentrations equal to the ATP added to the reaction. However, optimal
rates and yields in RecA-mediated DNA strand exchange require an
additional 6-8 mM of "free" Mg2+ (4-6).
Some of this magnesium ion is associated with the DNA, but the DNA
concentration in most experiments is on the order of a few
(1-20) µM in total nucleotides (and backbone
phosphate) and thus could not complex more than a small fraction of the
available magnesium ion. Lower magnesium concentrations, more
stoichiometric with the added ATP, are sufficient for primary DNA
binding. Some homologous DNA pairing occurs in the presence of ATPS,
an analog that is not appreciably hydrolyzed, but the higher magnesium
ion concentrations are required for the generation of long hybrid DNA
products with ATP (7). The extra Mg2+ is contributing
something significant to the reaction, but the effects have not been explained.
The question is intriguing, since the free magnesium ion requirements for RecA protein-mediated DNA strand exchange in vitro appear to exceed what is available in vivo. Mg2+ is present at about 100 mM in a bacterial cell (8-10), but almost all of this is bound up in ribosomes and DNA. The level of free magnesium ion is thought to be on the order of 1 or 2 mM at most (11). Based on in vitro data, the RecA protein should be almost inactive in the cell, although the crowding effect of the in vivo environment may moderate the effects of low available Mg2+ (12). There is a decreased requirement for magnesium ion in the presence of volume-occupying agents such as polyethylene glycol or polyvinyl alcohol, utilized to approximate the crowded environment of the cell, suggesting that an active conformation of RecA protein can be stabilized by either excess magnesium ion or the presence of crowding reagents at low magnesium (12).
The RecA protein has a small C-terminal domain extending from residue 270 to the protein terminus at residue 352, the function of which has not been fully explored (1, 13). The last 24 amino acid residues of the RecA protein are disordered in the published RecA protein crystal structures (14-16). We refer to this region as the RecA C terminus (as opposed to the entire C-terminal domain). Within this region, 7 of 25 amino acid residues are negatively charged. In addition to a general lack of structural information about the RecA C terminus, there has been little indication that this part of the protein has functional significance. Several C-terminal deletion mutants of the E. coli RecA protein have been characterized. Deletion from the C terminus of either 25 amino acid residues (17), or a fragment making up about 15% of the RecA polypeptide (18), resulted in faster nucleation, leading to filament formation on dsDNA. A proposal was advanced that the effect could be attributed to the elimination of electrostatic repulsion between the negatively charged residues in the C terminus and the phosphates in the DNA (17, 18). Both C-terminal deletion mutants promoted DNA strand exchange under at least some conditions, and the larger deletion exhibited an enhanced DNA strand exchange in the absence of single-stranded DNA-binding protein (SSB) (17, 18). The larger deletion exhibited ATPase and ssDNA binding activities similar to wild-type (18). The 25-residue deletion bound to ssDNA more tightly in some assays (17). A 25-residue C-terminal deletion of the RecA protein of Proteus mirabilis also exhibited improved binding to dsDNA (19).
Additional C-terminal deletions of RecA protein have also been constructed and studied, but without detailed biochemical characterization. A construct that removes most of the C-terminal domain (75 residues), when expressed with wild-type RecA, interferes with recombinational DNA repair and increases UV sensitivity slightly (20). A 17-residue C-terminal deletion mutant was shown not to affect UV resistance, induction of the SOS response, recombination, or Weigle reactivation when expressed on its own (21). A small effect on conjugational recombination was observed when the same mutant and wild-type proteins were both present in vivo (21). Removal of 18 residues from the C terminus resulted in a substantial conformational change in RecA filaments bound to dsDNA, suggesting an allosteric relationship between the C terminus and the RecA core domain (22).
In the previous paper (23), we characterized a set of RecA C-terminal
deletion mutant proteins designed to systematically test the role of
the acidic amino acids located in the C terminus of the RecA protein.
Removal of the last 13 (RecAC13), 17 (RecA
C17), or 25 amino acid
residues (RecA
C25) increased the rate of binding to dsDNA. The
C-terminal deletions also produced a profound effect on the pH
dependence of RecA protein-promoted DNA strand exchange reactions
relative to the wild-type protein. Ionizable groups in the C-terminal
region and others elsewhere in the protein appear to contribute to the
pH reaction profile.
In the present study, we show that the negatively charged C terminus of
the RecA protein has a modulating function on the DNA strand exchange
activity of RecA protein. In the absence of free magnesium ion, the C
terminus locks the protein in a conformation in which the initiation of
DNA strand exchange is inhibited. When the free magnesium ion
concentration is increased to 6-8 mM, the DNA strand
exchange function of RecA is activated. The results suggest that
magnesium ion interacts directly with RecA protein, altering the
conformation of the C terminus.
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EXPERIMENTAL PROCEDURES |
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Enzymes and Biochemicals--
The wild-type E. coli
RecA, RecAC6, RecA
C13, and RecA
C17 proteins were purified as
described (23). A plasmid containing the recA E343K mutant
(pEAW166) was constructed using PCR site-directed mutagenesis. The RecA
E343K point mutant protein was expressed and purified as described for
the wild-type RecA protein (23). The concentrations of the purified
RecA proteins were determined from the absorbance at 280 nm using the
extinction coefficient of 2.23 × 104
M
1 cm
1 (24). E. coli
SSB was purchased from Sigma. The concentration of the purified
SSB protein was determined from the absorbance at 280 nm using the
extinction coefficient of 2.83 × 104
M
1 cm
1 (25). Unless otherwise
noted, all reagents were purchased from Fisher. ATP
S was purchased
from Roche Molecular Biochemicals. Dithiothreitol was obtained from
Research Organics. Phosphoenolpyruvate, pyruvate kinase, ATP,
bromphenol blue, and NADH were purchased from Sigma. Restriction
endonucleases EcoRI, PstI, and SmaI
were obtained from New England Biolabs.
Bacteriophage M13 DNA Substrates--
Circular single-stranded
and supercoiled circular duplex DNAs from bacteriophage M13mp8 (7229 bp) were prepared using previously described methods (26-28). Except
where specifically noted otherwise, full-length linear duplex DNA was
generated by the digestion of M13mp8 supercoiled bacteriophage DNA with
the EcoRI restriction endonuclease, using conditions
suggested by the enzyme supplier. The digested DNA was extracted with
phenol/chloroform/isoamyl alcohol (25:24:1), followed by ethanol
precipitation. The concentrations of ssDNA and dsDNA solutions were
determined by absorbance at 260 nm, using 36 and 50 µg
ml1 A260
1,
respectively, as conversion factors. All DNA concentrations are given
in µM nucleotides.
DNA Three-strand Exchange Reactions Promoted by the Wild-type and
Deletion Mutant Proteins--
Three-strand exchange reactions were
carried out in 25 mM Tris-OAc buffer (80% cation) or 25 mM MES buffer (33% anion) (reaction pH 7.3 and 6.0, respectively, after the addition of all reaction components), 1 mM dithiothreitol, 5% (w/v) glycerol, 3 mM
potassium glutamate, and the indicated concentration of
Mg(OAc)2. Reactions also contained an ATP regeneration
system of 10 units/ml pyruvate kinase and 3.1 mM
phosphoenolpyruvate. All incubations are carried out at 37 °C. The
wild-type RecA, RecAC6, RecA
C13, or RecA
C17 proteins (6.7 µM) were preincubated with 20 µM M13mp8
circular ssDNA for 10 min. SSB protein (2 µM) and the
indicated amount of ATP were then added, followed by another 10-min
incubation. The reactions were initiated by the addition of M13mp8
linear dsDNA to 20 µM. A 10-µl aliquot was removed to
use as a zero time point, the reaction was incubated, and at the
indicated time points 10-µl aliquots were removed and the reaction
was stopped by the addition of 5 µl of a solution containing 60 mM EDTA, 6% SDS, 25% (w/v) glycerol, and 0.2% bromphenol
blue. Samples were subjected to electrophoresis at 10-20 mA in 0.8%
agarose gels with 1× TAE buffer (40 mM Tris-OAc, 80%
cation, and 1 mM EDTA), stained with ethidium bromide, and
exposed to ultraviolet light. Gel images were captured with a digital
CCD camera utilizing GelExpert software (Nucleotech). DNA bands were
quantitated with the software package TotalLab version 1.10 from Phoretix.
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RESULTS |
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Experimental Design--
The purpose of this study was to examine
the magnesium ion dependence of DNA strand exchange reactions promoted
by RecA C-terminal deletion mutants. We focused on the RecAC6,
RecA
C13, and RecA
C17 proteins described in the previous paper
(23). Under the standard reaction conditions for DNA strand exchange
promoted by wild-type RecA protein, the C-terminal deletion mutants
promote a significantly slower reaction (23). We set out to find
reaction conditions that improve the efficiency of the mutant
protein-catalyzed DNA strand exchange in an attempt to understand the
role of the negatively charged C terminus in the RecA-promoted DNA
strand exchange reaction.
The RecA C-terminal Deletion Mutants Require Less Magnesium Ion for
Optimal Strand Exchange than the Wild-type RecA Protein--
In an
attempt to find the optimal strand exchange conditions for the
C-terminal deletion mutants, magnesium titrations were carried out. DNA
strand exchange reactions were carried out in Tris-OAc buffer (reaction
pH 7.3) at 3 mM ATP and magnesium ion concentrations from 0 to 40 mM (Fig. 2). For each
protein, the final extent of strand exchange increases to an optimum
and then decreases as the concentration of magnesium ion is increased. For the wild-type RecA protein, this optimum occurs at about 10 mM magnesium ion, in line with many results published over
a period of two decades (4-6). Significantly, the deletion lacking 17 amino acids (RecAC17) exhibits optimal activity at magnesium ion
concentrations that are now roughly equivalent to the ATP concentration. Higher magnesium ion concentrations are inhibitory. The
optimal magnesium ion concentrations observed for the RecA
C6 and
RecA
C13 mutants fall between the other two, so that the magnesium ion requirements for the reaction decline as more of the C terminus is
removed. When reactions are compared at their respective optima, strand
exchange reactions progress to similar extents for all of the wild-type
and mutant RecA proteins.
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Just as the magnesium ion concentration needed for an optimal reaction declines as the C terminus of RecA is truncated, the magnesium ion concentrations needed to see inhibition of the strand exchange reaction also decline. At magnesium ion concentrations above 15 mM, strand exchange products by the wild-type RecA protein include large DNA complexes that do not readily enter the gel. These large complexes are protein-dependent (data not shown), are not resolved in 120 min, and appear at successively lower magnesium ion concentrations for the C-terminal deletion mutants.
The yield of DNA strand exchange products produced by each protein
after 60 min, at magnesium ion concentrations between 0 and 10 mM was quantitated (Fig. 3).
The products in this experiment include the bands corresponding to the
complex species in the well, joint molecules, and nicked circular
products of DNA strand exchange. These were totaled and divided by the
amount of all dsDNA (the above bands plus the linear dsDNA substrate)
in the lane. The bands at the well were included in the quantitation because they are protein-dependent and because they are
needed to account for all of the DNA in the lane. The magnesium ion
concentration required for maximum conversion of substrate DNA into
these products is ~10 mM for wild-type RecA, as has been
reported by several laboratories (4-6). As amino acid residues are
removed from the carboxyl terminus, the mutant proteins promote an
optimal DNA strand exchange reaction at progressively lower
concentrations of magnesium ion (Fig. 3).
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The RecAC17 Deletion Mutant Promotes an Optimal DNA Strand
Exchange Reaction When the Magnesium Ion Concentration Is Approximately
Equal to the ATP Concentration--
In order to determine whether the
RecA
C17 deletion mutant's optimal strand exchange conditions were
dependent on the ATP concentration, strand exchange reactions were
carried out at several ATP concentrations (1, 3, and 6 mM),
with the reaction extent examined as a function of magnesium acetate
concentration. At either 1 or 6 mM ATP (Fig.
4), the deletion mutant promotes an optimal strand exchange reaction at a magnesium level approximately equal to the ATP. Conversely, at 1 mM ATP, wild-type RecA
protein still requires 10 mM magnesium ion and requires an
even higher magnesium ion concentration when 6 mM ATP is
present. The reactions were quantitated such that only the nicked
circular final product of strand exchange was determined as a
percentage of all the duplex DNA species present (Fig.
5). It is clear that the wild-type RecA protein requires excess magnesium, above what is needed to complex with
ATP. As the ATP concentration increases, the concentration of magnesium
ion needed for an optimal reaction remains at 6-8 mM above
the ATP concentration. When the C-terminal 17 amino acids are removed
from the protein, the optimal DNA strand exchange reaction is observed
at a magnesium ion concentration that closely parallels the
concentration of ATP.
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A 3' Overhang on the dsDNA Substrate Enhances the Wild-type RecA
Protein-promoted DNA Strand Exchange Reaction at Low Magnesium Ion
Concentrations--
One of the results in Fig. 2 conflicts with a
recently published result from our laboratory (29), in which the
wild-type RecA protein promoted the formation of significant levels of
nicked circular product in DNA strand exchange reactions throughout a range of 1-11 mM Mg(OAc)2 (although the
optimal reaction was still seen with 11 mM
Mg2+, the reaction was significant but much reduced at 1 mM). As in many of the current trials, the ATP
concentration was 3 mM. This result appeared to contradict
not only the results above, but also other published results indicating
that wild-type RecA protein does not promote DNA strand exchange
reactions at low magnesium ion concentrations (7). We investigated this
apparent inconsistency in results. The DNA strand exchange reaction
results in Fig. 6 address this issue. The
DNA substrates utilized in the Rice et al. study were
derived from X174 bacteriophage, and the linear dsDNA substrate was
generated by digestion of circular dsDNA with the PstI
endonuclease (29). This treatment generates 3' overhangs at the DNA
ends, which are distinct from the 5' overhangs in the duplex DNA
substrates used in Figs. 2-5. To determine whether the overhangs play
a role in the reaction, we carried out DNA strand exchange reactions
with M13mp8 linear dsDNA substrates generated by circular dsDNA
digestion with the restriction enzyme PstI, EcoRI, or SmaI to generate 3' overhangs, 5'
overhangs, or blunt ends, respectively (Fig. 6). PstI
and EcoRI both leave 4-nucleotide overhangs. At 10 mM magnesium ion, the results of wild-type RecA reactions
using the different linear dsDNA ends are virtually indistinguishable,
whereas at 3 mM magnesium ion, appreciable amounts of the
nicked circular final product of strand exchange can be seen only when
3' overhangs are utilized. We note that the wild-type mediated reaction
with the EcoRI-cleaved DNA (5' overhangs) did result in more
reaction intermediates than the blunt-ended DNA, although they were not
converted to quantitatable final products. The 5' overhangs would be
complementary to the circular ssDNA, and thus enhance DNA pairing, at
the distal end of the linear duplex. The RecA
C17 protein appears to
not require any particular end, regardless of the magnesium ion
concentration.
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The RecAC17 Deletion Mutant Requires Low Magnesium Ion for Joint
Molecule Formation with ATP
S--
DNA strand exchange (with long
DNA substrates) generally does not proceed past the formation of joint
molecules when ATP is not hydrolyzed. However, the initiation of DNA
pairing in the presence of ATP
S should occur in a manner similar to
the more extended DNA strand exchange. As is the case for the DNA
strand exchange reaction with ATP, the optimal conditions for RecA
protein-promoted formation of joint molecule intermediates in the
presence of ATP
S include magnesium ion concentrations in significant
excess relative to the ATP
S that is present (30). We examined the
effect of DNA overhangs on both wild-type RecA and RecA
C17-promoted
DNA strand exchange reactions. The linear dsDNA substrates described above for Fig. 6 were used, with 3 mM ATP
S and either 3 or 10 mM Mg(OAc)2 (Fig.
7). At a magnesium ion concentration of
10 mM, joint molecules formed by wild-type RecA, using the
different linear dsDNA ends, are virtually indistinguishable. However,
at 3 mM Mg(OAc)2, appreciable amounts of joint
molecule products can be seen only when 3' or 5' overhangs (not blunt
ends) are utilized. The overall result is that a short single-stranded
overhang is needed for reactions to be initiated with the wild-type
protein in the absence of free magnesium ion. The reaction seen with 5' overhangs is consistent with the absence of polarity in the initial (ATP hydrolysis-independent) pairing reactions promoted by RecA protein. Intermediates produced on the distal end of the linear duplex
DNA (see Fig. 1) would be eliminated if ATP were hydrolyzed, as in Fig.
6.
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In contrast, RecAC17 appears to not require any particular end at 3 mM magnesium, but it is unable to form joint molecules at
10 mM Mg(OAc)2. The absence of observable joint
molecules in this reaction at the higher concentration of magnesium
ion, although a more complete DNA strand exchange reaction (albeit
somewhat suboptimal) is seen under these conditions, is taken up under "Discussion."
RecAC17 Protein Is Unable to Promote Complete DNA Strand
Exchange Reactions with ATP or Promote Joint Molecule Formation with
ATP
S at pH 6, Even at Low Magnesium Ion Concentrations--
In an
accompanying paper (23) characterizing the various C-terminal deletion
mutants of the RecA protein, we showed that the mutants were deficient
in their ability to generate nicked circular products in DNA strand
exchange reactions carried out at pH 6. In light of the results above
showing that the optimal magnesium ion concentration for the RecA
C17
protein-promoted reaction is ~3 mM, we tested the lower
magnesium ion concentrations in DNA strand exchange reactions with ATP
or ATP
S at pH 6 (Fig. 8). The
RecA
C17 protein is unable to promote a complete strand exchange
reaction with ATP or promote joint molecule formation with ATP
S at
pH 6, even at 3 mM magnesium ion. Thus, the effects of
magnesium ion are distinct from the pH effects noted elsewhere (23), at
least by this criterion. However, the wild-type RecA reaction at low
magnesium is somewhat stimulated by the decreased pH.
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The RecA E343K Point Mutant Requires Less Magnesium Ion for Optimal
Strand Exchange than the Wild-type RecA Protein--
To begin to
determine whether the negatively charged residues of the RecA C
terminus are contributing to the requirement for excess magnesium in
wild-type RecA-mediated DNA strand exchange reactions, we constructed a
mutant that replaces the acidic glutamate residue at position 343 with
a basic lysine residue (E343K). Position 343 lies in the protein
segment that is deleted in the RecAC13 mutant but is present in the
RecA
C6 truncation mutant (see Fig. 2). DNA strand exchange reactions
were carried out with wild-type RecA and the RecA E343K mutant at 3 mM ATP and magnesium ion concentrations from 1 to 15 mM (Fig. 9). The E343K mutant
promotes the strand exchange reaction at lower magnesium ion
concentrations than the wild-type protein. Using the criterion
described for the deletion mutants above, it appears that the optimal
reaction occurs for the E343K mutant at about 5-8 mM
Mg2+, similar to that of RecA
C6 protein. This experiment
was carried out twice with consistent results. Converting one of the
Glu residues in the C terminus to a positively charged Lys residue thus
has an effect similar to the deletion of 6 residues from the C
terminus.
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DISCUSSION |
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The primary conclusion of this study is that the C terminus of the
RecA protein modulates the protein's DNA strand exchange activity. The
last 17 amino acid residues are responsible for the observed
requirement for excess magnesium ion, above that necessary to form
complexes with ATP, in a wild-type RecA protein-promoted DNA strand
exchange reaction in vitro (31-34). Removal of these residues eliminates the measurable requirement for the excess Mg2+. These results strongly suggest that the RecA C
terminus has a regulatory role in RecA protein activity. At low
magnesium ion concentrations, the C terminus inhibits access of duplex
DNA ends to the RecA nucleoprotein filament, although single strands
(i.e. single strand extensions of duplex DNAs) can
facilitate a limited DNA pairing reaction. At least in
vitro, the protein undergoes a general activation for DNA strand
exchange in the presence of excess magnesium ion. Since the excess
Mg2+ is unnecessary for the RecAC17 deletion mutant, we
infer that a conformational change involving the C terminus is a key
part of the Mg2+-mediated activation process.
The primary structure of the C terminus offers ample opportunities for
interactions with magnesium ion. Contributions to the magnesium ion
interaction could be made by (a) one or more of the residues
Glu347, Glu350, and Asp351 (removed
in RecAC6), (b) one or more of the residues
Asp340, Asp341, and Glu343 (removed
in RecA
C13), and (c) residue Asp336 (removed
in RecA
C17). We note that the C-terminal 17 amino acid residues of
RecA protein also include three serine and threonine residues. There is
ample precedent that these could also be involved in magnesium ion
coordination (35, 36). One of these is removed in each of the 6-, 13-, and 17-amino acid residue deletions. However, the key Mg2+
binding site need not be in the C terminus; the present data do not
demonstrate Mg2+ binding at the C terminus, only that
Mg2+ affects the conformation of the C terminus so as to
bring about activation for DNA strand exchange. Egelman and colleagues
have demonstrated that the identity of the nucleotide bound at the distant ATP binding site can have a large impact on the conformation of
the C-terminal domain of RecA (37, 38). Similarly, the binding of
Mg2+ to a site in the core or elsewhere in the C-terminal
domain could cause a conformational shift that might affect the
positioning of the C terminus. In an accompanying paper (39), we
demonstrate that high Mg2+ levels have a significant effect on some
activities of the RecA
C17 protein.
The results are generally consistent with a C-terminal region that acts
as a protein flap to modulate access of the duplex DNA substrate to the
filament groove. This flap might act as a structural barrier, moved out
of the way via the proposed conformational change mediated by the
interaction of magnesium ion. Alternatively, the flap could act
somewhat indirectly, mediating conformational changes in other parts of
the protein that activate RecA. An allosteric effect of the C terminus
on the conformation of the RecA protein core domain has previously been
documented by Egelman and colleagues (22, 37). The wild-type RecA
protein is deficient in homologous pairing of duplex DNA at low
magnesium in the presence of ATP. Homologous pairing can be stimulated
at low magnesium if either the last 17 residues of the protein are
removed, as described above, or volume-occupying reagents are added to
the reaction (12). Additionally, the initiation of DNA pairing can be
stimulated, to a much lesser degree, if (a) ATPS is used
instead of ATP, (b) there is a single strand overhang
present on the duplex DNA substrate, or (c) the pH is
lowered to 6. Together, the results indicate that the C terminus is
inhibitory to DNA pairing and strand exchange at low Mg2+ concentrations.
We propose that the state of RecA protein, bound to ssDNA at low Mg2+ (or Mg2+ levels commensurate with the ATP present) be designated Ac. The status of RecA on single-stranded DNA in the presence of ATP has historically been referred to as activated (40-42), hence the "A." The state present at low Mg2+ is relatively closed to interaction with incoming duplex DNA, hence the "c."
A change in state is needed to facilitate DNA pairing and strand exchange. In most studies carried out with the E. coli RecA protein, the change is brought about by adding Mg2+ in excess to the ATP concentration. We propose that the resulting state be denoted Ao, using the "o" to denote that the protein is now open to DNA pairing with duplex DNA regardless of end structure. It is unlikely that these states reflect single, distinct conformations in the presence of ATP. Egelman and colleagues (22, 37, 38) have amply demonstrated that the hydrolysis of ATP can bring about a variety of conformational changes, particularly in the C-terminal domain. Each of the proposed states can hydrolyze ATP with nearly equal facility, as described below.
The noticeable enhancement of DNA pairing by short single-strand overhangs under the low Mg2+ conditions suggests that the barrier to DNA pairing is much reduced for ssDNA relative to duplex DNA. Even short single strands at the end of a duplex can provide sufficient stabilization of the initial pairing process to overcome the barrier and allow some DNA pairing to occur.
Magnesium ion could affect DNA strand exchange at many stages, but it is the stages after the formation of RecA-ssDNA nucleoprotein filaments that seem to be most affected. Previous data have suggested that the wild-type RecA protein does not require excess magnesium ion to bind to ssDNA. The rates of ssDNA-dependent ATP hydrolysis catalyzed by the wild-type RecA protein with 1 mM magnesium ion and 0.5 or 1 mM ATP and no SSB protein are close to the rates measured at 10 mM magnesium in the presence of SSB protein (43, 44). Given the similarities in ssDNA binding and ATP hydrolysis, the structural differences between the proposed Ac and Ao states may be subtle. The key functional distinction is that the low Mg2+ conditions do not support initiation of a robust DNA strand exchange reaction.
There is additional evidence in the literature that Mg2+
concentrations affect RecA conformation. RecA-ssDNA nucleoprotein
filaments have been observed in the electron microscope in the presence of 1 mM magnesium. Notably, the contour lengths of these
filaments were measured to be 116-120% relative to duplex DNA in the
presence of 1 mM ATPS (7) and 137% relative to duplex
DNA in the presence of 1.3 mM ATP (45), which translates to
less filament extension than the >150% extension observed when
the filaments are formed with 10 mM magnesium ion (23, 46,
47). It is possible that this reduced filament extension seen at low
Mg2+ concentrations is a result of an inhibitory
conformation of the C terminus. Elevated Mg2+ levels also
result in the formation of RecA filament bundles observable by electron
microscopy (46, 48, 49). Finally, with elevated Mg2+
levels, RecA protein better resists displacement by SSB (50).
The RecAC17 protein exhibits a substantially altered pH reaction
profile in DNA strand exchange reactions (23). We have determined that
this pH dependence is not affected by magnesium, since, at pH 6, the
RecA
C17 protein is deficient in homologous pairing with ATP
S and
in the formation of nicked circular product with ATP at low (Fig. 8) or
high magnesium levels (23).
Combining the data from this study and the previous paper (23) allows us to refine the protein flap model for the function of the C-terminal domain of RecA protein. We propose that the C-terminal amino acid residues of wild-type RecA protein are inhibitory not only to the primary binding of RecA protein to establish a filament on dsDNA (as previously proposed) (17, 18) but also to DNA pairing of duplex DNA with a single strand bound within a RecA filament. These may be quite distinct processes, since the barrier to direct binding of RecA protein monomers to a duplex DNA should be different from the binding of a duplex DNA to a RecA filament already formed on a single strand. The barrier to direct binding of RecA protein to dsDNA is overcome at pH 6 with wild-type protein, presumably reflecting the protonation of one or more residues in the C terminus, but is not overcome by excess magnesium ion at neutral pH values. This again indicates that the pH- and magnesium ion-mediated changes in protein state are to some degree distinct, albeit both involve the C terminus in some way.
Kowalczykowski and colleagues (18) previously proposed that the C-terminal domain had a role in modulating DNA assimilation and strand exchange. However, their more substantial deletion led to an enhancement of DNA strand exchange (with no SSB present) under conditions similar to those in which the deletions studied here decrease the efficiency of strand exchange.
Shibata and colleagues (51) have put forth a model for homologous
pairing in which the "gateway" for dsDNA binding to the presynaptic
filament lies in the filament groove that is made up of, on one side,
the C-terminal domain of the RecA protein. Mutations of some of the
many basic residues in this cleft have been shown to abolish homologous
pairing. The residues involved include Arg243,
Lys245 (52, 53), Lys286, and Lys302
(51) (Fig. 10). These residues all lie
within 20 Å of the last residue of the RecA protein seen in the
apoenzyme crystal structures, Leu328 (14, 15). There are 24 C-terminal residues (including the seven negative charges that we have
removed in the current study) that were disordered in those structures.
It is possible that the negative charges of the C terminus can form
salt bridges with basic residues in this cleft, thereby restricting the
access of dsDNA to the presynaptic filament. These interactions may be
part of a network of surface salt bridges. A network would help to explain the gradual reduction in the requirement for magnesium seen
with the progressive removal in the negative charges of the C terminus.
Each salt bridge disruption could affect the strength of the next. Such
a network would explain the effects of the RecA E343K mutant, which
exhibits a behavior similar to RecAC6 although that mutation is not
in the region removed with RecA
C6. Magnesium could act by disrupting
those salt bridges, enabling homologous pairing. In this way, the
C-terminal tail of the RecA protein could form a flap that regulates
accessibility to the nucleoprotein filament. The coupling of protein
surface salt bridge disruption to DNA binding is a common mechanism
used by DNA-binding proteins and has been reviewed recently (54).
|
As mentioned in the Introduction, the levels of free Mg2+
available in the cell are insufficient to bring about the activation seen in vitro. It is possible that the molecular crowding in
the cell substitutes for the effects of high Mg2+ levels
(12). Alternatively, another molecule may replace Mg2+ in
the cell as an activating agent.
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ACKNOWLEDGEMENTS |
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We thank Jim Keck, Ruth Saecker, and Tom Record for useful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM32335.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
University of Wisconsin, 433 Babcock Dr., Madison, WI 53706-1544. Tel.:
608-262-1181; Fax: 608-265-2603; E-mail:
cox@biochem.wisc.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M212916200
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ABBREVIATIONS |
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The abbreviations used are:
ssDNA, single-stranded DNA;
dsDNA, double-stranded DNA;
ATPS, adenosine
5'-O-(thiotriphosphate);
SSB, single-stranded DNA-binding
protein;
MES, 4-morpholineethanesulfonic acid.
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