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INTRODUCTION |
Escherichia coli RecF is a multifunctional protein,
with identified roles in recombinational DNA repair, homologous genetic recombination, and DNA replication. The phenotypes of recF mutants include decreased plasmid recombination (1, 2), moderate UV sensitivity
(3), attenuated SOS response (4, 5), and deficiency in the repair of
daughter strand gaps (6, 7). A role for RecF protein in replication was
suggested by the requirement for recF (and recR) in the resumption of
replication after the collapse of DNA replication forks (8) and by the
requirement of recF for cell viability in a priA mutant background (9). A link between recombination and replication is implied in one model
(10) which postulates that the in vivo role of RecF, in conjunction with RecO and RecR, is to divert damaged
ssDNA1 from the replication
process so that recombinational DNA repair can take place. In
vitro, RecF protein binds to both ssDNA and dsDNA and hydrolyzes
ATP (11-14). The RecF and RecR proteins, acting together, limit the
extension of RecA filaments beyond DNA gaps (15). All of the functions
of RecF may come together in recombinational DNA repair, providing a
useful paradigm within which to examine RecF protein activities (16,
17).
A detailed in vitro characterization of the RecF protein can
help in elucidating function. The DNA binding properties of RecF protein have been described in several reports. The binding to ssDNA
binding is ATP-independent (11, 12), although binding to dsDNA is
ATP-dependent (13, 14). RecF protein binds to dsDNA weakly
in the presence of ATP but binds much more stably when RecR is included
or in the presence of the nonhydrolyzable ATP analog, ATP
S (14). The
role of the ATP hydrolytic activity of RecF in dsDNA binding has not
been analyzed. The RecF protein contains a consensus nucleotide binding
fold (GXXGXGKT) in the N-terminal
portion, which is highly conserved among the known recF gene sequences.
The relevance of this nucleotide binding fold to the in vivo
function of RecF was suggested by the observation that a single amino
acid substitution within it (Lys to Arg at position 36) appears to be a
null allele (recF4101) (18, 19). Comparable mutations in the conserved
ATP binding sites of RecA (20), UvrB (21), and RAD3 (22) also produce
mutant phenotypes in vivo and result in mutant proteins that
bind but do not hydrolyze ATP. A further indication that the capacity
to hydrolyze ATP plays a role in RecF function is the recF4101 (also
RecF K36R, with a mutation in the ATP binding site) overexpression
phenotype. Overexpression of wild-type RecF renders cells inviable at
42 °C. However, cells overexpressing the RecF4101 protein are viable (23). In light of these findings, we have studied the role of ATP
hydrolysis in RecF protein function, focusing on DNA binding. We have
also examined the effects of RecR protein and the binding of RecF
protein to gap junctions in DNA.
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MATERIALS AND METHODS |
Enzymes and Reagents--
E. coli RecF and RecR
proteins were purified as described (14). The mutant RecF protein, RecF
K36R, was purified according to the procedure used for wild-type RecF
protein. All protein preparations were free of detectable endo- or
exonuclease activities. Protein concentration was determined by
absorbance at 280 nm using the extinction coefficients:
280 = 3.87 × 104
M
1 cm
1 for RecF protein and
RecF K36R protein (this work) and
280 = 5.60 × 103 M
1 cm
1 for RecR
protein (24). At no time during purification or storage was the RecF
protein subjected to conditions, as defined in this work, under which
the protein would aggregate and would be inactivated. The purified RecF
and RecF K36R proteins are shown in Fig.
1.

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Fig. 1.
Purified RecF protein. RecF and RecF
K36R proteins were purified using the RecF protocol described
previously (14). Numbers shown at the left of the
gel represent the molecular mass of the marker proteins (M) in
kDa.
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Restriction endonucleases were purchased from New England Biolabs. MES
and EPPS buffers, bovine serum albumin, ATP, glycerol, sodium chloride,
phosphocreatine, creatine phosphokinase, NADH, and phosphoenolpyruvate
were from Sigma. Oligonucleotides were synthesized by the University of
Wisconsin Biochemistry Department Synthesis Facility.
[
-32P]dideoxyATP (3000 Ci/mmol) was from Amersham
Pharmacia Biotech. ATP
S was purchased from Boehringer-Mannheim Biochemicals.
The extinction coefficient for native RecF protein was determined by a
published procedure (25), modified as described (26). The results of
four determinations were averaged to give an extinction coefficient of
native RecF as
280, M, native = 3.87 ± 0.12 × 104 M
1 cm
1. The
RecF concentrations determined using this method were in good agreement
with the concentration determined by the Bradford method. This same
extinction coefficient was used for the RecF K36R mutant.
DNA--
Supercoiled circular duplex
X174 RFI DNA was
purchased from Life Technologies, Inc. Linear
X174 dsDNA was
prepared by complete digestion by PstI. Circular ssDNA from
M13mp8.1037 was prepared as described (24). Bacteriophage M13mp8.1037
was derived from M13mp8, having a 1037-base pair insertion
(EcoRV-EcoRV fragment from the E. coli
galT gene) in the SmaI site. Linear dsDNA radiolabeled at
the 3'-end was generated using terminal transferase and
[
-32P]dideoxyATP. The concentrations of dsDNA and
ssDNA stock solutions were measured by absorbance at 260 nm, using 50 and 36 µg ml
1 A260
1,
respectively, as the conversion factors. DNA concentrations are
expressed in terms of total nucleotides. DNA was stored in TE (10 mM Tris-HCl, pH 7.5, plus 1 mM EDTA)
Gapped DNA with a precisely defined gap length was prepared essentially
as described (27). Gapped DNA having a 1037 base single-stranded gap
(GD1037) was prepared by annealing NaOH-denatured M13mp8
linearized by SmaI digestion to M13mp8.1037 (28) circular ssDNA. GD3329 was prepared by annealing the 4937-base pair
fragment of a SmaI-BspHI digest of M13mp8 to
M13mp8.1037 circular ssDNA. Thus GD1037 and
GD3329 have a common 8266 base M13mp8.1037 inner circle and
have different length linear DNAs annealed to it (7229 and 4937 bp,
respectively). As an additional purification step, to remove
contaminating M13mp8.1037 circular ssDNA and linear M13mp8 dsDNA after
hydroxylapatite chromatography, the gapped DNA was electrophoresed on a
0.8% SeaPlaque GTG-agarose gel, and the DNA band corresponding to
GD1037 or GD3329 was excised. The gel slice was
melted at 65 °C and extracted 1:1 with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1). The final preparation of DNA was precipitated twice with ethanol.
Construction of RecF Mutant RecF K36R--
To generate a plasmid
containing the mutant recF allele, recF4101, and thus permitting
overexpression of the RecF K36R protein, site-directed mutagenesis was
performed using an oligonucleotide similar to that used by Sandler
et al. (19) (5'-CAGCACGCTGGTACGGCCGCTGCCGTTGGCA-3') and
using pBLW20 as the template (14). The resulting plasmid encoded a
mutant recF gene with the lysine at position 36 changed to arginine.
This change was verified by sequencing, and the
XbaI-NcoI fragment containing this mutation was
then ligated back into pBLW20 to produce pBLW23. Overexpression of RecF
K36R protein was performed exactly as for wild-type RecF protein, by
transforming pBLW23 into the E. coli strain K38/pGP1-2
(29).
Reaction Conditions--
Unless stated otherwise, reactions were
performed in buffer A: 25 mM buffer (see below), 10 mM MgCl2, 50 mM sodium chloride, 5% (w/v) glycerol, 0.1 mM EDTA, and 0.1 mM
dithiothreitol. MES buffer was used for reactions at pH 5.7, 6.1, and
6.5. EPPS buffer was used for reactions at pH 6.7, 7.2, 7.6, 8.1, and
8.5. The reported pH of reaction mixtures reflect pH measurements (at
25 °C) of solutions containing all reaction components (except that storage buffers were substituted for proteins).
RecF Protein Aggregation Studies--
We observed that RecF
protein aggregates under certain conditions, causing an increase in
turbidity at 340 nm when introduced into reactions monitored
spectrophotometrically. Therefore, we used this signal as a measure of
RecF protein aggregation to determine reaction components that prevent
this effect. A Perkin-Elmer Lambda 7 double-beam recording
spectrophotometer equipped with two thermojacketed cuvette holders,
each capable of holding six cuvettes, was used for absorbance
measurements. The cell length and the band pass were 0.5 cm and 2 nm,
respectively. All components indicated except RecF and RecR were added
to a 400-µl reaction mixture containing buffer A (EPPS, pH 7.6, except as stated otherwise). This was preincubated in the
spectrophotometer at 37 C until the absorbance reading at 340 nm was
stable. Then RecF protein (0.5 µM) and, as indicated,
RecR protein (1.0 µM) were added, and the apparent absorbance (signal increase reflecting scattered light) was
automatically measured every 30 s. Relative RecF protein
aggregation was measured as an increase in the absorbance at 340 nm
because of turbidity. The average of duplicate experiments is reported.
The data is used comparatively, and no absolute measure of the degree
of RecF protein aggregation is either known or implied.
Electron Microscopy--
Reactions for observing RecF K36R
protein binding to linear dsDNA were performed in buffer A (EPPS, pH
7.2) including 3 µM
X174 linear dsDNA, 1 mM ATP, and 0.6 µM RecF K36R protein. The reactions for observing RecF protein binding to dsDNA in the presence of ATP
S were performed in buffer A (pH 8.1) including 1 µM
X174 linear dsDNA, 1 mM ATP
S, and
0.2 µM RecF protein. The pH studies of wild-type RecF
protein were performed in buffer A at the indicated pH including 1 µM
X174 linear dsDNA, 1 mM ATP or ATP
S,
and 0.2 µM RecF protein. In all experiments, after the
addition of RecF protein (or RecF K36R protein), the reactions were
incubated at 37 °C for 10 min. The samples were then spread
undiluted and without cross-linking for microscopy, as described
previously (14). The grids were washed in reaction buffer containing
15% glycerol and of the appropriate pH before staining and shadowing.
RecF ATPase Assays--
ATP hydrolysis by RecF protein was
measured by a coupled enzyme assay. A Perkin-Elmer Lambda 7 was used
for absorbance measurements in this assay. The regeneration of ATP from
ADP and phosphoenolpyruvate coupled to the oxidation of NADH can be
followed by the decrease in absorbance at 340 nm (the absorption
maximum, which provides a level of sensitivity sufficient for the
measurement of the relatively weak ATPase activity of RecF protein).
Rates of ATP hydrolysis were measured at 37 °C at the following pH
values: 5.7, 6.1, 6.5, 6.7, 7.2, 7.6, 8.1, and 8.5. Reaction mixtures
(400 ml) contained buffer A of the indicated pH, 20 µM
X174 linear dsDNA, and 3 mM ATP. An ATP regenerating
system (1.5 mM phosphoenolpyruvate, 4.5 units
ml
1, and 3 mM potassium glutamate) and a
coupling system (0.6 mM NADH and 4.5 units
ml
1 lactate dehydrogenase) were also included. The basal
level of the decrease in absorbance at 340 nm was measured for 45 min
prior to the addition of RecF protein. The rate of ATP hydrolysis
catalyzed by RecF protein was calculated by subtracting this basal
level from the decrease in absorbance at 340 nm measured for 45 min after the addition of RecF protein (2.5 µM). At each pH,
the average of duplicate experiments is reported. Duplicate
measurements of rates greater than 0.4 µM
min
1 varied by no more than 7%, and those below 0.4 µM min
1 varied by no more than 15%.
Gel Shift Experiments--
Agarose gel assays were carried out
to detect complexes between RecF protein and linear dsDNA, as described
previously (14). Challenge reactions were performed by preincubating
RecF protein with 32P-labeled dsDNA in the presence of
either ATP or ATP
S prior to challenging with unlabeled dsDNA to
determine how the stability of RecF·dsDNA complexes was affected by
nucleotide cofactor and other parameters. RecF protein (1.33 µM) was preincubated with 32P-labeled linear
dsDNA (20 µM, as indicated in appropriate figure legends)
in a 25-µl reaction mixture containing either ATP or ATP
S in
buffer A (EPPS, pH 8.1) at 37 °C. After 10 min, unlabeled linear
dsDNA (10 or 100 µM, as indicated) was added to the
reaction (or TE for the control reaction). Portions of the reaction
were stopped at the indicated times by the addition of EDTA (10 mM). A gel loading buffer (25 mM Tris-HCl, pH
7.5, 50% glycerol, and 0.02% bromphenol blue) was then added, and
samples were electrophoresed on a 0.7% agarose gel at 3 V/cm with
recirculation of the running buffer (20 mM Tris-HCl, pH
7.5, 4 mM sodium acetate, 0.1 mM EDTA) at room
temperature. The gel was then dried, and the DNA was detected by autoradiography.
A modified assay patterned after a published procedure (30) was used to
detect complexes between RecF and linear dsDNA or gapped DNA.
Competition reactions in which one DNA substrate was preincubated with
RecF prior to challenging with the second DNA substrate were used to
detect any preferential binding. The reaction buffer contained 20 mM Tris-HCl (61% cation), 50 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA, and 5%
(w/v) glycerol. Reactions containing 15 µM DNA
(GD1037, M13mp8.1037 linear dsDNA, or both as indicated), 0.3 µM RecF (giving one RecF per 50 nucleotides), and 1 mM ATP
S were incubated at 37 °C for 30 min. The final
pH of the mixture was 7.59 at 25 °C. For the challenge reactions,
RecF was preincubated with one of the DNA cofactors for 10 min at
37 °C, followed by addition of the second DNA with the incubation
continued for 30 min. A gel loading buffer (25 mM Tris-HCl,
pH 7.5, 50% (w/v) glycerol, and 0.025% bromphenol blue) was added to
each reaction, and the samples were immediately electrophoresed on a
0.8% agarose gel at 2.5 V/cm in 25 mM Tris-HCl, pH 7.5, 4 mM sodium acetate, and 1 mM EDTA with
recirculation of the buffer. The DNA was then detected by ethidium
bromide staining.
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RESULTS |
RecF Protein Undergoes Aggregation in Vitro--
In the presence
of ATP, the RecF protein appears to exist as a monomer in solution. In
gel filtration trials carried out on columns equilibrated with buffer
containing 1 mM ATP and 100 mM NaCl, RecF
protein (40.5 kDa) consistently migrates slightly slower than the
22-kDa RecR protein (14).2
Under these conditions, Umezu and Kolodner (31) reported that RecR
existed as a dimer, a result we have confirmed,2 so that
the slower migration of RecF protein is most consistent with a
monomeric species.
Our own observations and hints in some published reports suggested that
the activity of RecF proteins may be affected by instability and/or
insolubility in vitro. Madiraju and Clark (12) noted that
incubation of prediluted RecF protein for 30 min prior to its addition
to a reaction mixture resulted in total loss of DNA binding activity.
We observed that RecF protein loaded onto a gel filtration column does
not elute unless the column is equilibrated and developed in buffer
containing either ATP or high salt (1 M NaCl) (14).
Subsequently, we noticed that solutions containing RecF protein became
visibly cloudy with what appeared to be precipitated or aggregated
protein under a variety of conditions. We do not know whether the
cloudy appearance of the solutions is because of precipitated protein
or simply large aggregates. We generally employ the term
"aggregation" throughout this paper, without implying any
particular physical effect.
To follow up on these observations, we have used changes in turbidity
as a measure of RecF protein aggregation or precipitation in
vitro. As applied, this method is not intended to represent a
quantitative measurement of the fraction of RecF protein aggregated. Instead, it was used to compare relative levels of RecF protein aggregation under different conditions and to identify solution conditions where this could be avoided.
RecF protein added to a reaction mixture containing no ATP or DNA at
37 °C aggregated almost immediately, as indicated by a large
increase in turbidity (Fig.
2A). With RecF protein concentrations above 1 µM, the aggregation was manifested by a visible cloudy precipitate in the solution. The effect was
temperature-dependent, as RecF protein was completely
stable at 4 °C but aggregated shortly after a shift to 37 C (Fig.
2A). Exclusion of Mg2+ from the buffer delayed
but did not eliminate this phenomenon (data not shown). To determine
whether RecF protein was irreversibly inactivated by aggregation, the
resulting white precipitate was collected by centrifugation,
resuspended in storage buffer, and assayed for DNA binding activity. No
activity was detected (data not shown). We do not know whether the
protein is partially denatured and thereby inactive, or if it is simply
present as large aggregates that are inaccessible to substrates. In
either case, to date we have been unable to restore RecF protein to a
form capable of ATP hydrolysis or DNA binding once the protein has come
out of solution.

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Fig. 2.
Aggregation of RecF protein in
vitro. The aggregation of RecF following its addition
to a reaction mixture was monitored by increases in apparent absorption
caused by turbidity at 340 nm. Reactions were carried out as described
under "Materials and Methods" and contained buffer A (EPPS, pH 7.6)
and the indicated reaction components. After the addition of RecF (0.5 µM) and, as indicated, RecR (1.0 µM),
absorbance readings were automatically taken every 30 s.
A, RecF aggregation occurred rapidly when added to buffer A
at 37 °C. RecF added to buffer A equilibrated at 4 °C remained in
solution until the temperature was shifted to 37 °C. The temperature
shift required about 15 min to complete in this experiment. Aggregation
detectable by turbidity increases is not evident until the temperature
approaches 37 °C. B, aggregation of RecF protein in
buffer A at 37 °C in the presence of the indicated reaction
components. The concentration of ATP and ATP S was 1 mM.
Linear X174 dsDNA and M13mp8.1037 circular ssDNA were both used at
10 µM. Where indicated, 30% (v/v) glycerol, 2 mg/ml BSA,
and 150 mM NaCl were also added.
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Because diluted RecF protein has been shown to be active for DNA
binding and ATP hydrolysis under a variety of solution conditions, the
reaction components that prevent or delay the aggregation of RecF
protein were determined. As shown in Fig. 2B, aggregation is
not affected by the addition of dsDNA but is decreased by ATP. Increasing the ATP concentration from 1 to 3 mM resulted in
markedly less aggregation (data not shown). When present together,
dsDNA and ATP slow the precipitation even more, suggesting a
synergistic effect. Like ATP, ATP
S had only a small effect on
aggregation. However, ATP
S in combination with dsDNA greatly reduced
the observed turbidity change. These are conditions under which RecF
protein is stably bound to dsDNA (14). Even though RecF protein remains in solution much longer when bound to DNA, it does slowly precipitate even under these conditions (Fig. 2B).
In the absence of ATP, RecR protein had no effect on RecF protein
aggregation either in the presence or absence of dsDNA. RecR protein
also had no effect in the presence of ATP when dsDNA was not also
present (data not shown). However, the combination of the RecR protein,
ATP, and dsDNA greatly reduced the turbidity increase (Fig.
2B). These are again conditions under which RecF protein
(and RecR) are stably bound to the DNA (14, 15). Interestingly, although the ssDNA binding activity of RecF has been reported to be
ATP-independent, aggregation of RecF protein as measured by changes in
turbidity was reduced by ssDNA in the presence of ATP (Fig.
2B) but not in the absence of ATP (data not shown).
The above studies indicate that RecF protein tends to aggregate and
becomes inactive when it is not bound to DNA. In an attempt to find
solution conditions which might stabilize RecF protein in the absence
of DNA, we tested a variety of salts, detergents, and stabilizing
agents for their ability to prevent RecF protein aggregation. We found
that a combination of 30% glycerol, 2 mg/ml BSA, and 150 mM NaCl significantly reduced RecF protein precipitation in
the absence of ATP or DNA. As indicated by a lack of significant increases in turbidity, RecF protein was completely soluble and unaggregated for at least 2 h when these stabilization components were combined with ATP
S and dsDNA (Fig. 2B). Although we
have not fully characterized the effect of these conditions on RecF activities in vitro, they do not appear to significantly
affect the dsDNA binding activity of RecF as measured by an agarose gel shift assay or its ATPase activity (data not shown).
The Role of ATP Hydrolysis in dsDNA Binding, a Mutant RecF Protein
(RecF K36R) That Cannot Hydrolyze ATP Binds Tightly to
dsDNA--
Using electron microscopy, two previous observations
inferred that ATP hydrolysis by RecF protein leads to its dissociation from dsDNA (14). First, RecF protein binds tightly to dsDNA in the
presence of a nonhydrolyzable ATP analog, ATP
S. Second, much less
RecF protein is bound in the presence of ATP, which is hydrolyzed by
RecF protein. To verify that an inability to hydrolyze ATP blocks the
dissociation of RecF protein from dsDNA, we generated and purified a
RecF mutant protein that alters the ATP binding site (RecF K36R). The
mutation is identical to the recF4101 mutant described by Sandler
et al. (19). RecF K36R does not hydrolyze ATP (14).
RecF K36R protein was assayed for ATPase activity in six separate
trials carried out over a period of 5 months. Four of these utilized
the spectrophotometric assay described under "Materials and
Methods," while the other two used the thin layer chromatography method (14). In two of the trials, multiple reactions were run side by
side with and without the RecR protein, which stimulates ATP hydrolysis
by the wild-type RecF protein (14). Reactions were carried out at pH
7.2 under standard reaction conditions, except that four of the trials
included the optimal stabilizing conditions of 30% glycerol, 100 mM NaCl, 2 mg/ml BSA, and 3 mM ATP. ATP
hydrolysis was not detected at any level in any of these trials (data
not shown). We conclude that at least under these conditions, the RecF
K36R mutant protein does not hydrolyze ATP.
As shown in Fig. 3, RecF K36R protein
binds to and coats linear dsDNA in the presence of ATP. In a count of
98 molecules selected at random from two separate experiments, 97 or
99% were coated with protein. The molecule in Fig. 3B is
typical of this population, with molecules varying somewhat in the
number and lengths of small discontinuities in the protein coat. The
DNA bound with mutant protein appears identical to dsDNA coated with
wild-type RecF protein in the presence of ATP
S. In the presence of
ATP, RecF K36R protein also retards the migration of dsDNA in an
agarose gel to the same extent as wild-type RecF protein in the
presence of ATP
S (data not shown; see Fig. 5 for an example of the
result obtained with wild-type protein and ATP
S). Unlike the
wild-type protein, the binding of the mutant RecF K36R protein to dsDNA was reduced in the presence of ATP
S. This was true regardless of
whether the stabilizing reaction conditions were employed. The reduced
binding was manifested by protein-free or nearly protein-free DNA
observed by electron microscopy in three separate trials, in contrast
to the coated DNA seen when ATP was used in Fig. 3B. In a
count of 182 individual DNA molecules (linear
X174 dsDNA) from three
separate experiments, 158 or 87% had no bound protein. Small dots or
short tracts appearing to be bound RecF K36R protein were seen on 24 molecules, all of these from one of the three samples. No molecules
were completely coated with protein. In three gel retardation trials
(data not shown), the reactions with ATP
S produced a variable
smearing of the DNA band (data not shown) as opposed to the sharp
retarded band seen with ATP. In the complete absence of nucleotide
cofactor, little or no binding to dsDNA was observed with the wild-type
RecF protein (12, 14). This also proved to be the case for the RecF
K36R mutant protein, as measured by either electron microscopy or gel
retardation (two trials each; data not shown). In the electron
microscope, 107 of 114 molecules counted in two separate experiments,
or 94%, had no bound RecF K36R protein in the absence of nucleotide
cofactors. A few DNA molecules in both experiments (7 total) had small
tracts that appeared to be bound protein. Inasmuch as the presence of ATP clearly facilitates the binding of RecF K36R protein to dsDNA, we
inferred that the mutant protein was able to bind ATP as well as
DNA.

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Fig. 3.
Comparison of linear dsDNA bound with RecF
K36R in the presence of ATP and with wild-type RecF in the presence of
ATP S. Binding reactions were carried out
as described under "Materials and Methods." After a 10-min
incubation at 37 °C, the samples were spread for microscopy without
cross-linking or dilution. A, linear X174 dsDNA without
RecF added. B, linear X174 dsDNA (3 µM) was
incubated with RecF K36R (0.6 µM) in the presence of ATP.
C, linear X174 dsDNA (1 µM) was incubated
with wild-type RecF (0.2 µM) in the presence of
ATP S.
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Wild-type RecF protein binds stably to dsDNA in the presence of ATP
at low pH--
To further correlate the relationship between RecF ATP
hydrolysis and dissociation from dsDNA, we studied the effect of pH on
these two processes using wild-type RecF protein. The pH profile of the
ATPase activity of RecF in the presence of linear dsDNA is shown in
Fig. 4A. At pH 5.7, no ATP
hydrolysis by RecF protein was detected. With increasing pH, the ATPase
activity increased, reaching a maximum around pH 8.1.

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Fig. 4.
Dependence of RecF-mediated ATP hydrolysis
and dsDNA binding in the presence of ATP on pH. Reactions were
performed at 37 °C in buffer A at the indicated pH, as described
under "Materials and Methods." A, RecF ATP hydrolysis
rates as a function of pH. Reactions contained 20 µM
X174 linear dsDNA, 3 mM ATP, and 2.5 µM
RecF. The pH values of the individual reactions were 5.7, 6.1, 6.5, 6.7, 7.2, 7.6, 8.1, and 8.5. B, effect of pH on RecF
binding to dsDNA in the presence of ATP. Reactions contained 1 µM X174 linear dsDNA, 1 mM ATP, and 0.2 µM RecF and were carried out at pH 5.6 (A),
6.5 (B), 7.2 (C), and 8.1 (D). The
molecules shown were chosen to be representative of their respective
samples. At pH 5.7, the DNA is essentially fully coated with protein.
The complexes have a much thicker appearance than free dsDNA. The
protein coat has no visible fine structure under these preparative
conditions. At pH 6.5, there is visibly less bound protein. Short
portions of the DNA appear to be fully coated, but more usually there
are a high diversity of individual protein blobs. At pH 7.2, there are
no fully coated regions, and only individual protein blobs are
observed. At pH 8.1, very few protein blobs are observed. Many DNA
molecules have no detectable bound protein. There was no indication of
cooperative protein binding in any of the samples.
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The rates of ATP hydrolysis were monitored using a coupled
spectrophotometric assay as a decrease in absorbance (because of NADH
oxidation) with time at 340 nm. These measurements could be affected by
RecF aggregation, which would cause a slower decrease in measured
absorption because of increases in turbidity (which we measured at the
same wavelength, albeit the reactions did not contain NADH), as well as
the accompanying loss of RecF activity. For the reactions performed
below pH 7.5, no significant change in rate was detected over the
45-min time course used to calculate the steady state ATP hydrolytic
rate, suggesting that no measurable aggregation of RecF protein
occurred. However, there was an apparent decline in the rate with time
in the reactions performed at the higher pHs. In agreement with the
precipitation studies above, this most likely reflects an increase in
absorbance because of precipitation, which nullifies some of the
decrease in absorbance brought about by NADH oxidation in the coupled
reaction system. This became more noticeable with increasing pH. As a
result, the rates for reactions above pH 7.5 were calculated from the
first 30 min of time points after the addition of RecF protein to
minimize any effects of aggregation.
We previously reported a turnover number of 0.2 min
1 at
pH 7.0, a result obtained with a different ATPase assay based on
thin-layer chromatography (14). The rate for pH 7 derived from the data in Fig. 4A was 0.64 min
1. We attribute much of
the increase to the effects of the 5% glycerol present in the new
experiments, which greatly reduces aggregation and its accompanying
loss of activity. The highest rate observed, at pH 8.1, was 0.97 min
1. Three other trials were carried out over a period
of 4 months under conditions determined to stabilize the RecF protein
(5% or more glycerol plus high concentrations of ATP), two using the thin layer chromatography assay (14) and the other using the spectrophotometric assay. All were carried out at or near pH 7 with 1 mM or greater ATP. The kcat derived
from these trials varied from 0.5 to 0.58 min
1.
A pH titration of the dsDNA binding activity of RecF was carried out
using electron microscopy. The amount of RecF protein bound to linear
dsDNA in the presence of ATP
S was not affected by pH, within the
range of 5.7-8.1; incubation of dsDNA with saturating amounts of RecF
protein resulted in fully coated DNA at all pHs (data not shown). Fig.
4B shows the effect of pH on the binding of RecF protein to
dsDNA in the presence of ATP. In agreement with our earlier report
(14), very little RecF protein was bound to the dsDNA above neutral pH,
where the ATPase activity is greatest. However, at pH 5.7, where the
ATPase activity is suppressed, RecF protein bound and coated the dsDNA.
Thus, the extent of the binding of RecF protein to dsDNA is correlated
inversely with its ATP hydrolytic activity. Unlike at pH 7.6 (Fig.
2B), RecF protein exhibited little aggregation as reflected
by measured turbidity in the presence of ATP and dsDNA at pH 5.7 (data
not shown), presumably because it remains bound to the dsDNA.
Though the ATPase activity of RecF protein is relatively weak and
accumulation of ADP in a 10-min incubation is unlikely, we tested the
effect of including an ATP regeneration system on RecF protein binding
to dsDNA in the presence of ATP at pH 8.1. As determined by electron
microscopy, the extent of RecF binding to dsDNA was unchanged (data not shown).
RecF Protein Binds Stably to dsDNA in the Presence of
ATP
S--
The migration of linear dsDNA through an agarose gel is
retarded by RecF protein to a greater extent in the presence of ATP
S than with ATP (14). To determine whether less RecF protein is bound to
the dsDNA in the presence of ATP as a result of ATP hydrolysis-induced dissociation, we examined the stability of these RecF·dsDNA complexes over time using a challenge experiment. Radiolabeled linear dsDNA (20 µM) was preincubated with RecF protein (1.33 µM) in the presence of either ATP or ATP
S for 10 min
prior to challenging with excess unlabeled linear dsDNA (100 µM). Aliquots of the reaction were stopped with EDTA at 5 and 10 min after the challenge and run on an agarose gel. RecF protein
dissociated rapidly from the labeled dsDNA when bound in the presence
of ATP. Within 5 min after the challenge DNA was added, the retardation
in the migration of the labeled DNA was reduced substantially, almost
to the point of running even with the unbound DNA marker
(Fig. 5, lane 1 versus lanes 2 and 3). In contrast,
the migration of dsDNA bound by RecF protein in the presence of ATP
S
was retarded almost to the same extent before and after the challenge
(Fig. 5, lane 4 versus lanes 5 and
6), indicating little dissociation of RecF protein. Thus, ATP hydrolysis correlates with the dissociation of RecF from the DNA.

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Fig. 5.
Dissociation of RecF from dsDNA in presence
of ATP versus ATP S. A
challenge experiment was used to measure dissociation of RecF bound to
dsDNA. The reactions were carried out as described under "Materials
and Methods." RecF (1.33 µM) was preincubated with
32P-labeled X174 linear dsDNA (20 µM) in
the presence of either ATP or ATP S, as indicated, 10 min prior to
the addition of excess unlabeled X174 linear dsDNA (100 µM). RecR protein (2.66 µM) was included in
the reactions shown in lanes 7-9. Reactions were stopped by
the addition of EDTA after 5 and 10 min, and the extent of RecF binding
to the labeled dsDNA was determined using an agarose gel retardation
assay. M, X174 linear dsDNA markers.
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The RecR Protein Slows the Dissociation of RecF Protein from
dsDNA--
Addition of the RecR protein improves the binding of RecF
protein to dsDNA in the presence of ATP (14). To explore the molecular basis of this phenomenon, we examined the effect of RecR protein on
RecF dissociation from dsDNA (Fig. 5, lanes 7-9). The
presence of RecR protein does not prevent dissociation of RecF protein from the DNA, but the dissociation of the putative RecFR complexes are
markedly slowed relative to the dissociation of RecF alone (compare
lanes 7-9 to lanes 2 and 3).
RecF Protein or RecFR Complexes Do Not Exhibit a Preferential
Association with ssDNA-dsDNA Junctions in Gapped DNA--
A recent
report (30) indicates that in the presence of ATP
S, RecF protein has
a higher affinity for gapped DNA (gDNA) than for either dsDNA or ssDNA,
implying it might bind tightly to the ss-ds junction of the gDNA. In
contrast, a study of the effects of RecF and RecR proteins on the
assembly of RecA filaments on gapped DNA provided no indication that
RecFR complexes bind to the ss-ds junction (15). We wished to determine
whether RecF protein alone exhibits an enhanced affinity for gap
junctions that might be abolished by dATP/ATP hydrolysis and/or an
interaction with RecR protein.
As a direct assessment of the relative affinity of RecF protein for
gDNA versus dsDNA, gel shift DNA binding assays were carried out, patterned after the work by Hedge et al. (30) and done under essentially the same reaction conditions except for the inclusion
of 5% glycerol and the use of somewhat different DNA substrates.
Subsaturating amounts of RecF protein were incubated with either linear
dsDNA (end-labeled with 32P), GD1037, or both
in the presence of ATP
S (Fig. 6). To
visualize the location of both DNAs simultaneously, we stained the
agarose gel with ethidium bromide (as shown). The identity of each band was verified separately by exposing the dried gel to x-ray film (data
not shown). With either DNA substrate alone, increasing the RecF
protein concentration from 180 nM (1 RecF per 83 nucleotides) to 300 nM (1 RecF per 50 nucleotides) resulted
in a readily discerned decline in the mobility of the DNA in the gel
(Fig. 6, lanes 3-6). When both DNAs were included in the
experiment at equal concentrations, RecF protein (1 RecF per 50 total
nucleotides) impeded the migration of each of them to a similar extent
(lane 7), reflecting the binding of RecF protein to both
DNAs. In this experiment, the amount of available dsDNA in the two
types of DNA molecules is quite similar, differing only by the absence
of one strand over about 15% of the length of the gDNA.

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Fig. 6.
RecF exhibits no preferential binding to
gapped DNA over dsDNA. Reactions were carried out as described
under "Materials and Methods," with 15 µM DNA
(GD1037, M13mp8.1037 linear dsDNA, or both as indicated),
RecF (at the indicated concentration), and 1 mM ATP S. At
the end of the incubation, samples were electrophoresed on a 0.8%
agarose gel. The gel was loaded as it was running such that the
lanes at right have migrated for a somewhat
shorter time than the lanes at left. The
dashed line is drawn between points 1 cm below the linear
dsDNA marker in lane 1 and a similar marker in a
lane to the right of lane 10 (not
shown), to provide a measure of the degree of retardation caused from
left to right as a result of this loading effect. The DNA was detected
by ethidium bromide staining. Controls: lane 1, linear dsDNA
marker; lane 2, GD1037 marker; lane
3, 0.3 µM RecF with linear dsDNA (1 RecF:50
nucleotides total DNA); lane 4, 0.18 µM RecF
with linear dsDNA (1:83); lane 5, 0.3 µM RecF
with GD1037 (1:50); lane 6, 0.18 µM RecF with GD1037 (1:83). To
determine whether RecF preferentially binds to gapped DNA, RecF (at 300 nM or 1:50 nucleotides of total DNA) was incubated with
both DNAs for 30 min at 37 °C (lane 7), or was preincubated with one
of the DNAs 10 min prior to challenging with the other DNA for an
additional 30 min (linear dsDNA before GD1037 in
lanes 8 and 9; GD1037 before linear dsDNA in
lane 10).
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Challenge experiments were also carried out. Linear dsDNA was
preincubated with RecF protein (300 nM, or 1 RecF per 50 nucleotides) for 10 min prior to challenging with an equal amount of
GD1037, followed by incubation for an additional 30 min
(lanes 8 and 9). The linear dsDNA was shifted
almost to the full extent of the control (lane 3) with
linear dsDNA alone, while the GD1037 was shifted very
little. In addition, the GD1037 band (the upper
band in lanes 8 and 9) did not have the
somewhat smeared appearance evident in the bound DNA bands in
lanes 3-6. Thus, once bound to the linear dsDNA, very
little RecF protein was seen to move to the GD1037.
Similarly, most of the RecF protein initially bound to the
GD1037 remained there when challenged by linear dsDNA
(lane 10). These data indicate that RecF protein binds with
apparently equal facility to the dsDNA or to the dsDNA portion of gDNA.
The experiments were carried out 5 times with consistent results. Elimination of the glycerol (5%) did not affect the result (data not
shown). We found no evidence for a preferential binding of RecF protein
to gap junctions.
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DISCUSSION |
Our primary conclusion is that ATP hydrolysis by RecF protein
leads to its dissociation from dsDNA. Stable binding is observed in the
presence of ATP
S, which is not hydrolyzed by the RecF protein, and
at low pHs where ATP hydrolysis is minimal. The RecF K36R mutant, which
does not hydrolyze ATP, also binds stably to dsDNA when ATP is present.
The dissociation of RecF protein from dsDNA is slowed by the RecR protein.
We also show that RecF protein aggregates in vitro at
37 °C, becoming both insoluble and inactive under conditions in
which it is not bound to DNA. The aggregation is slowed by the addition of glycerol, NaCl, and BSA to the reaction, or by incubation at relatively low temperatures. If DNA and an appropriate nucleoside triphosphate are present, aggregation is minimized. The aggregation of
RecF at 37 °C may contribute to the formation of inclusion bodies in
cells in which RecF is highly expressed (14). Soluble RecF protein is
obtained in greater yield when the expressing cells are grown at
reduced temperatures. The aggregation and accompanying inactivation of
RecF protein in vitro may complicate the interpretation of
any experiments involving RecF protein done under conditions where it
is not bound to DNA.
We previously showed that RecF binding to dsDNA in the presence of ATP
is limited (14). Here we show that inhibiting RecF ATP hydrolysis,
either by replacing ATP with ATP
S, by using a RecF mutant that is
unable to hydrolyze ATP, or by lowering the pH such that the hydrolytic
activity of wild-type RecF is blocked, leads to stable binding of RecF
protein to dsDNA and prevents its dissociation. In addition, RecF
protein dissociation from dsDNA in the presence of ATP but not ATP
S
was observed directly using a DNA challenge experiment.
The precedents for such a role of ATP hydrolysis in DNA binding
proteins that bind ATP are numerous. The ADP-bound forms of the RecA
protein (24, 32), Rep helicase (33), and UvrA (34) protein all have
lower affinity for their DNA substrates than ATP-bound forms, resulting
in an increased tendency to dissociate following ATP hydrolysis. The
functional significance of dissociation from DNA in each of these cases
is specific to the individual protein and the role they play in DNA metabolism.
The RecF K36R mutant protein binds to dsDNA in the presence of ATP, but
the binding is much reduced when ATP
S is substituted for ATP. We do
not have an explanation for the effects of ATP
S. The analogous
mutation in the ATP binding site of the RecA protein, RecA K72R,
results in a protein that does not hydrolyze NTPs and functions in
certain RecA activities. However, this mutant RecA functions with
either dATP or ATP
S, but not with ATP (20, 35).
The binding of RecF protein to both ssDNA and dsDNA is readily
demonstrated (11-15). However, we have been unable to confirm a report
that RecF protein binds preferentially to gap junctions (30). Complexes
of the RecF and RecR proteins appear to bind randomly to the dsDNA
portion of gapped DNA molecules, where they halt RecA filament
extension (15). In the present study, direct comparisons of the binding
of RecF protein alone to dsDNA or gapped DNA failed to reveal
significant differences between the two.
The molecular function of ATP hydrolysis and subsequent dissociation
from dsDNA by RecF protein in vivo is not entirely clear. One obvious function could be the recycling of the protein. However, there are many other possibilities. RecF may interact with a number of
recombination and replication proteins in vivo. The RecF and RecR proteins interact in vitro (14), and together they
limit the extension of RecA protein filaments beyond single-stranded DNA gaps (15). Therefore, one function of RecF protein may be to
modulate RecA filament assembly. The weak ATPase activity of RecF
protein could also play some role in regulating the interface between
recombination and replication systems during recombinational DNA repair
(17). A complete understanding of the role of RecF-mediated ATP
hydrolysis must await analysis of the effects of additional recombination and replication proteins on RecF and of the effects of
RecF on these proteins.