(Received for publication, November 11, 1996, and in revised form, February 3, 1997)
From the Department of Chemistry and Biochemistry,
and § Program in Molecular and Cell Biology, University of
Maryland, College Park, Maryland 20742
We have expressed the RecD subunit of the RecBCD
enzyme from Escherichia coli as a fusion protein with a
31-amino acid NH2-terminal extension including 6 consecutive histidine residues (HisRecD). The overexpressed fusion
protein can be purified in urea-denatured form by metal chelate
affinity chromatography. The mixture of renatured HisRecD protein and
the RecB and RecC proteins has a high level of
ATP-dependent nuclease activity with either single- or
double-stranded DNA, enhanced DNA unwinding activity, enhanced ATP
hydrolysis activity in the presence of a small DNA oligomer cosubstrate, and -cutting activity. These are all
characteristics of the RecBCD holoenzyme. The HisRecD protein
by itself hydrolyzes ATP in the presence of high concentrations of
single-stranded DNA (polydeoxythymidine). The activity is unstable at
37 °C, but is measurable at room temperature (about 23 °C). The
HisRecD has very little ATPase activity in the presence of a much
shorter single-stranded DNA (oligodeoxy(thymidine)12).
HisRecD hydrolyzes ATP more efficiently than GTP and UTP, and has very
little activity with CTP. We also purified a fusion protein containing
a Lys to Gln mutation in the putative ATP-binding site of RecD. This
mutant protein has no ATPase activity, indicating that the observed ATP hydrolysis activity is intrinsic to the RecD protein itself.
The RecBCD enzyme from Escherichia coli is an important
enzyme in the DNA metabolism of the cell, acting in homologous
recombination, resistance to UV irradiation and chemical DNA damaging
agents, and degradation of foreign DNA (reviewed in Refs. 1-3). The
enzyme catalyzes several reactions in vitro. ATP hydrolysis
enables it to unwind double-stranded DNA (4-6), and the enzyme is a
potent nuclease on double-stranded DNA in the presence of ATP and
excess magnesium ion (7, 8). The double-strand nuclease activity of
RecBCD is suppressed when it encounters a sequence (5
-GCTGGTGG) in
the DNA (9-12). The enzyme continues to unwind the DNA past the
sequence (11) and the unwound DNA is a substrate for recombination catalyzed by the RecA protein (13-15). RecBCD enzyme is also an ATP-stimulated nuclease with single-stranded DNA, and single-stranded DNA stimulates ATP hydrolysis (7, 8).
The first preparations of the RecBCD enzyme were thought to contain only the proteins encoded by the recB and recC genes (8, 16-19). However, one experiment indicated the possible existence of an additional component, essential for enzymatic activity, which could be separated from both the RecB and RecC proteins by treating the enzyme with high salt concentrations (20). A later study showed that this was a third protein subunit, called RecD, encoded by a gene (recD) adjacent to the recB gene (21).
The function of the RecD subunit in catalysis by RecBCD enzyme is not
clear, although it is implicated in several activities of the enzyme.
The identification of the RecD subunit was made possible by the fact
that it is required for high levels of nuclease activity with single-
or double-stranded DNA (21, 22). Amino acid sequence analysis shows
that RecD and RecB contain consensus ATP-binding motifs as well as
other sequences similar to those in a family of DNA helicases (23). A
RecBCD enzyme with a mutation in the ATP-binding site of RecB retains
single-stranded DNA-dependent ATPase activity, which we
concluded is catalyzed by the wild-type RecD subunit in that enzyme
(24). The enzyme with a Lys to Gln mutation in the putative ATP-binding
site of the RecD subunit (the RecBCD-K177Q enzyme) retained all the
activities for which we tested (ATPase, nuclease, and helicase),
although each was reduced in rate and other properties (25, 26). The
effect of sequences on the nuclease activity has led to the
proposal that
causes inactivation of, and perhaps ejection of, the
RecD subunit (2, 27-30). This is consistent with the fact that RecBC is a helicase but lacks significant nuclease activity (31-33), and
that recombination in recD
cells is not
affected by
sequences (22).
Purification of the RecD protein has been difficult and has led to
little insight into its function in the reactions catalyzed by RecBCD.
No catalytic activity was found to co-purify with the RecD subunit
isolated from the RecBCD holoenzyme (20, 21). The RecD protein was
overexpressed and purified from inclusion bodies, and the isolated
subunit restored nuclease activity and -specific cleavage to RecBC,
but no activity was found in RecD alone (32). The difficulty of
obtaining RecD protein in native form led us to prepare RecD fused to
an NH2-terminal peptide containing six consecutive
histidine residues. The protein (HisRecD) can be purified by metal
chelate affinity chromatography in either native or denatured form, and
reconstituted with the RecB and RecC proteins to obtain high levels of
ATP-dependent nuclease activity, and other activities
characteristic of the RecBCD enzyme. The HisRecD protein alone
catalyzes ATP hydrolysis in the presence of high concentrations of
single-stranded DNA.
Materials
Isopropyl -D-thiogalactopyranoside, guanidinium
hydrochloride, and DTT1 were from U. S. Biochemical Corp.; Triton X-100 was from J. T. Baker; urea
(electrophoresis grade) was from Fisher Scientific Corp.; and HEPES and
imidazole were from Sigma. Ribonucleoside triphosphates (100 mM solutions), pd(T)12,
pd(T)25-30, and poly(dT) (average size: 221 nt) were
purchased from Pharmacia. [
-32P]ATP (3000 Ci/mmol),
[
-32P]CTP, [
-32P]GTP, and
[
-32P]UTP (each 800 Ci/mmol) were purchased from
DuPont Corp. Oligodeoxynucleotides for polymerase chain reactions were
purchased from DNA International, Lake Oswego, OR.
Restriction enzymes, Taq DNA polymerase, and T4 DNA ligase
were from Promega Corp., U. S. Biochemicals Corp., or New England Biolabs. Bovine serum albumin was from New England Biolabs. Exonuclease I was purchased from U. S. Biochemicals Corp. RecJ exonuclease was a
gift from Prof. Richard Kolodner, Harvard University. E. coli single-stranded DNA-binding protein was purified as described (26, 34). E. coli strain V186 (recBCD; Ref. 35) and
pBR322-
FH plasmid DNA (11) were gifts from Dr. Gerald Smith, Fred
Hutchinson Cancer Research Center, Seattle. 3H-Labeled
pTZpB700 plasmid DNA (5001 base pairs, constructed by us for unrelated
experiments) was prepared as described (25).
RecBCD enzyme was
purified as described (36), and the concentration was determined from
the absorbance at 280 nm, using 280 = 4 × 105 M
1 cm
1 (6). The
RecB and RecC proteins were purified and quantitated, using
280 = 1.7 × 105
M
1 cm
1 (RecB), and
280 = 2 × 105
M
1 cm
1 (RecC) as described (32,
33). The concentrations of each enzyme or subunit given under
"Results" and in the figure legends are those based on the
absorbance readings, unless stated otherwise. However, each enzyme
preparation apparently contained some inactive enzyme. The nuclease
activity of RecBCD was maximal with about 2 enzyme molecules per DNA
end (measured as in Ref. 25, with 2 and 4 nM ends). Thus
the RecBCD enzyme was about 50% active. The rate of ATP hydrolysis by
RecBC (prepared by mixing 1 µM RecB and 1 µM RecC) was maximal at about 4 RecBC per DNA end
(measured as in Ref. 37), indicating that only about 25% of the RecB
and/or RecC was active.
The His-tagged RecD protein (HisRecD) was expressed
using the vector pTrcHisB (Invitrogen Corp.). The recD gene
was transferred to the vector as follows. A 712-base pair fragment from
the plasmid pPvSm19, which contains the recD gene (36), was
amplified by polymerase chain reaction. The downstream primer annealed
across the BspEI site (bold) within the gene (primer number
1: 5-GGCATCTTCCGGAATG) and the upstream primer annealed at
the 5
-end of the gene (primer number 2:
5
-GGAAGATCT). Primer
number 2 is partially complementary to the recD gene
(underlined) and introduces a BglII site upstream of the
gene (bold). The amplified 712-base pair fragment was digested with
BspEI and BglII and ligated into pPvSm19 cleaved
with the same enzymes (the BglII site in pPvSm19 is within a
portion of the recB gene contained in this plasmid). The
recD gene was removed from this plasmid by cleavage with
BglII and BamHI (BamHI cleaves
downstream of the 3
-end of the recD gene in pPvSm19), and
ligated into pTrcHisB cleaved with the same enzymes, to produce
pHisRecD. The recD fragment is in the correct orientation
for expression when the BglII sticky ends are joined to the
compatible BamHI ends.
The resulting plasmid (pHisRecD, 6.6 kilobase pairs) encodes the RecD
protein fused to a 31-amino acid leader peptide (3.4 kDa) containing
six consecutive histidine residues. The protein is expressed from a
trc promoter and a ribosome-binding site within the vector
sequence. The RecD amino acid sequence was altered slightly during the
subcloning procedure, since the first two residues in RecD, Met-Lys,
are changed to a single Asp in the fusion protein. The 5 terminus of
the fusion gene was sequenced to confirm the structure of the junction
between the vector and recD gene sequences.
The NH2-terminal leader peptide encoded by the vector also includes a sequence recognized by the protease enterokinase (-Asp-Asp-Asp-Asp-Lys-). However, we have been unable to cleave the NH2-terminal peptide with enterokinase (Invitrogen Corp.), under a variety of reaction conditions, enterokinase concentrations, incubation times, etc. Consequently we have worked with the fusion protein itself, since it gives high levels of activity despite the additional peptide.
Construction of a Plasmid Expressing His-tagged RecD-K177Q Protein (pHisRecD-K177Q)The plasmid pPvSm19-DK177Q, encoding a Lys to Gln mutation in the putative ATP-binding site of RecD (36), was digested with BsmI, and the 442-base pair fragment containing the mutation was ligated into pHisRecD cleaved with the same enzyme. The presence of the mutation in the resulting plasmid, pHisRecD-K177Q, was also confirmed by DNA sequencing.
Methods
Purification and Renaturation of His-tagged RecD ProteinThe HisRecD protein was expressed in E. coli
strain JM109 (recA
recBCD+) or V186 (
recBCD) transformed
with pHisRecD. Cells were grown in 25 ml of LB broth (38) at 37 °C.
Isopropyl
-D-thiogalactopyranoside (1 mM)
was added when the A650 = 0.4, and growth was
continued for 4 h. The cells were harvested and resuspended in
either denaturing binding buffer (20 mM sodium phosphate,
pH 7.8, 0.5 M NaCl, 6 M guanidinium
hydrochloride) or native binding buffer (20 mM sodium phosphate, pH 7.8, 0.5 M NaCl). For the denatured
preparation, the resuspended cells were rocked gently for 10 min at
room temperature, and then the lysates were cleared by centrifugation
for 10 min at 17,000 × g. The lysate was applied to a
2-ml nickel-containing column (ProBond resin, Invitrogen Corp.) in
denaturing binding buffer. The column was washed in 20 mM
sodium phosphate, pH 6.0, 8 M urea, 0.5 M NaCl,
and then the same mixture at pH 5.3. The HisRecD fusion protein was
eluted in denaturing elution buffer (20 mM sodium
phosphate, pH 4.0, 8 M urea, 0.5 M NaCl). Its
concentration was determined from the absorbance at 280 nM,
using
280 = 48,500 M
1
cm
1, calculated for HisRecD (the NH2-terminal
leader peptide contains a single tyrosine residue, and no tryptophans).
The typical yield was about 0.5 mg from 25 ml of culture.
For preparation under native conditions, lysozyme (0.1 mg/ml) was added to the resuspended cells, followed by incubation for 10 min at 37 °C, 5 min on ice, and then the lysate was cleared by centrifugation as above. The lysate was applied to a 2-ml ProBond column which was washed with 20 mM sodium phosphate, pH 7.8, 0.5 M NaCl, then with 20 mM sodium phosphate, pH 6.0, 0.5 M NaCl, and finally the bound protein was eluted in a gradient of 50-500 mM imidazole in 20 mM sodium phosphate, pH 6.0, 0.5 M NaCl.
The denatured HisRecD protein was renatured by first diluting with denaturing elution buffer to less than 50 µg/ml, and then dialyzing in a collodion membrane (25-kDa cut-off; Schleicher & Schuell) at 4 °C against renaturation buffer (10 mM Tris-HCl, pH 8.0, 1 mM DTT, 0.1% Triton X-100, 0.5 M NaCl, 20% glycerol). The denatured HisRecD protein was stable when stored at 4 °C. However, we have not found conditions under which the renatured HisRecD can be stored without substantial loss of reconstitution activity. Thus, HisRecD was renatured as needed.
Reconstitution of HisRecD with RecBCThe reconstitution
method used for most experiments was as follows: the renatured HisRecD
protein (in renaturation buffer) was mixed with RecC (in RecBCD
dilution buffer: 10 mM potassium phosphate, pH 7.0, 0.1 mM EDTA, 0.1 mM DTT, 0.4 mg/ml bovine serum albumin, 20% glycerol), and placed at room temperature for 4 h. RecB (in RecBCD dilution buffer) was then added and incubation was
continued overnight at room temperature. The final reconstitution mixture usually contained 80 nM RecB and RecC and 300 nM HisRecD, in a 1:1 mixture of renaturation buffer and
RecBCD dilution buffer. A few reconstitutions were done by adding
HisRecD to RecBC and incubating for 2-4 h at room temperature. The
reconstituted RecBC(HisRecD) enzyme is relatively stable. The enzyme
lost only 20% of its nuclease activity on double-stranded DNA after 2 months at 20 °C, in this solution.
Native gel electrophoresis was carried out on 5% polyacrylamide gels (30:1, acrylamide:bis-acrylamide) prepared in imidazole/HEPES buffer (43 mM imidazole, 35 mM HEPES, pH 7.4) containing 10 mM MgCl2. The gels were pre-run at 5.5 V/cm for 30 min before protein samples were loaded, and then the samples were run at 3.5 V/cm. The gels were silver-stained with Rapid-Ag-Stain (ICN Biomedicals).
RecBCD Enzyme Assay Methods
The standard reaction conditions we used for these experiments were 43 mM imidazole, 35 mM HEPES buffer, pH 7.4, 10 mM MgCl2, 0.67 mM DTT, at 37 °C, unless indicated otherwise. The reconstituted RecBC(HisRecD) prepared as above was diluted 10-50-fold with a 1:1 mixture of renaturation buffer and RecBCD dilution buffer, and then diluted 10-100-fold further into the reaction mixtures. Thus, the components of the renaturation and reconstitution buffers were also present in the reaction mixtures. The other enzymes used in these experiments (RecBCD holoenzyme, RecBC, and HisRecD alone) were also diluted with the same buffer mixture, so that all enzymes were compared under the same reaction conditions.
NucleaseNuclease activity was determined by measuring the production of trichloroacetic acid-soluble oligonucleotide fragments (36), using [3H]pTZpB700 DNA (25 µM nt), linearized by cleavage with PstI. Reactions with double-stranded DNA contained 50 µM ATP, while those with heat-denatured DNA contained 200 µM ATP.
Hydrolysis of Ribonucleoside TriphosphatesNucleoside
triphosphate hydrolysis was measured by thin layer chromatography, as
described (25), using - or
-32P-labeled
ribonucleoside triphosphate substrate. Polyethyleneimine chromatography
plates (J. T. Baker) were developed in 1 M formic acid, 0.5 M LiCl2 for ATPase reactions, or 1 M formic acid, 1 M LiCl2 for CTP,
GTP, and UTP. The relative amounts of [
-32P]ATP and
[32P]Pi, or [
-32P]NTP and
[
-32P]NDP, on the developed plates were determined
using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
DNA unwinding was assayed by the conversion of [3H]pTZpB700 DNA (21 µM nt), linearized with PstI, to a form sensitive to exonuclease I and RecJ exonuclease, as described (37). Reaction mixtures contained 200 µM ATP, 10 mM MgCl2, and 8 µM E. coli single-stranded DNA-binding protein.
-Specific cleavage
reactions were done essentially as in Ref. 39 using pBR322-
FH (11),
containing two
sequences in opposite orientations. The plasmid was
cleaved with ClaI, treated with calf intestinal alkaline
phosphate, and 5
-end-labeled with polynucleotide kinase, and
[
-32P]ATP. Markers for
-specific cleavage were
prepared by cleaving the labeled, ClaI-cut DNA again with
AvaI.
The -cutting reactions contained 20 mM MOPS-KOH, pH 7, 5 mM ATP, 3.5 mM magnesium acetate, 5 µM E. coli single-stranded DNA-binding protein, 1 mM DTT, and ClaI-cut
[5
-32P]pBR322-
FH (3.1 nM DNA molecules;
26.7 µM nt), at 37 °C. Samples removed from the
reaction mixtures were quenched by adding 0.2 volumes of a mixture
containing 40% glycerol, 2.5% SDS, 0.1 M EDTA, 0.125%
bromphenol blue, and analyzed by electrophoresis on 1% agarose gels.
The radioactivity in the dried gel was visualized using the
PhosphorImager.
A protein of the expected size (slightly larger than RecD)
was found in lysates of cells transformed with pHisRecD, after induction with isopropyl -D-thiogalactopyranoside. We
purified the histidine-tagged RecD protein (HisRecD) under both native and denaturing conditions, following the procedures recommended by
Invitrogen Corp. The HisRecD could be highly purified in a single step
(Fig. 1; some preparations contained a small amount of
an impurity, probably the 21-kDa protein seen previously in His-tagged proteins purified from E. coli (40)).
The assay for RecD activity was the ability to restore
ATP-dependent nuclease activity to RecBC, on linear single-
and double-stranded DNA. The nuclease activities with both single- and
double-stranded DNA (Fig. 2) are completely dependent on
the presence of RecB, RecC, HisRecD protein, and ATP (not shown), as
expected for RecBCD enzyme activity (8, 21, 32). The reconstituted
enzyme had a high level of activity in this assay. The activity of the
reconstituted enzyme depends on: 1) the efficiency with which the three
protein subunits assemble to make RecBC(HisRecD), and 2) the fraction of renatured HisRecD protein which is active. We estimated both quantities by comparing the nuclease activity of the reconstituted enzyme to that of the RecBCD holoenzyme. The nuclease activity on
double-stranded DNA with RecBC(HisRecD) prepared by mixing RecB and
RecC (2 nM each) with a large excess of HisRecD (45 nM), followed by 10-fold dilution into the assay, was the
same as that of about 0.06-0.065 nM RecBCD in the assay
(not shown). After accounting for the presence of inactive RecBCD and
RecB/C protein (see "Experimental Procedures"), this suggests that
about 60-65% of the RecBC could be converted to RecBC(HisRecD): 65% = (0.065/2 nM active RecBCD)/(0.2/4 nM active
RecB/C) × 100. The reconstituted enzyme used for the experiment in
Fig. 2A was prepared by mixing HisRecD (290 nM)
with an excess of RecB and RecC (400 nM each). The activity
on double-stranded DNA, with 0.58 nM HisRecD, and 0.8 nM RecB and RecC in the assay mixture, was about the same as that of 0.17 nM RecBCD (Fig. 2A). This
suggests that at least (0.17/2)/0.58 ×100 = 15% of the HisRecD
is active, or 23%, if only 65% of RecB and RecC assemble to
RecBC(HisRecD). (Both calculations assume that the specific activity of
100% reconstituted RecBC(HisRecD) would be the same as that of
RecBCD.)
The nuclease activity appeared when the proteins were reconstituted at room temperature (about 23 °C), but little activity was found if the reconstitution was done on ice, under any conditions. The time required varied somewhat depending on the protein concentrations. Thus, the nuclease activity in a mixture of 3.5 nM RecBC and 45 nM HisRecD required about 2 h before reaching a maximal level. The rate of appearance of activity did not increase significantly if HisRecD was incubated overnight with either RecB or RecC and then the remaining subunit was added, as compared with adding HisRecD to RecBC. This suggests that the slow appearance of the nuclease activity is not due to slow assembly of HisRecD with only one of the other two proteins, since in that case overnight preincubation with that particular subunit would have allowed final assembly of the RecBC(HisRecD) complex to be rapid. (We did notice that slightly greater activity (about 2-fold) was obtained by adding the subunits sequentially (the method given under "Experimental Procedures") rather than incubating all three subunits together. Thus we used that procedure as the standard way to prepare RecBC(HisRecD) for further experiments.)
The HisRecD purified under native conditions had lower activity than
the renatured HisRecD, but greater activity was obtained when this
HisRecD protein was first dialyzed against renaturation buffer. This
protein was also less pure after the affinity chromatography step than
was the denatured HisRecD. We also purified HisRecD from V186 cells
(recBCD) and obtained nuclease activity when the protein
was added to RecB and RecC, indicating that the presence of the
wild-type recBCD genes in JM109 did not affect the HisRecD preparation from those cells. Most further experiments were done with
HisRecD purified under denaturing conditions from JM109.
We also tested several factors during renaturation for their effects on the yield of active HisRecD. Dialysis against renaturation buffer containing (NH4)2SO4 (0.5 M) worked about as well as NaCl, and both gave greater activity than dialysis against the same concentrations of KCl, NH4Cl, or potassium glutamate. Dialysis to lower the urea concentration in steps (4, 2, 1, 0 M) did not lead to significantly greater yield of activity. There was no effect of including ZnCl2 (0.1 mM; see Ref. 32) in the dialysis buffer. Lower activity was observed when: 1) renaturation was done at room temperature rather than 4 °C; 2) renaturation buffer contained 0.2 M NaCl rather than 0.5 M NaCl; 3) the renaturation buffer was MOPS, pH 6.5, rather than Tris-HCl, 8.0.
Native Polyacrylamide Gel ElectrophoresisThe nuclease
activities shown above strongly indicate that the HisRecD is bound to
RecBC to make a RecBC(HisRecD) complex. We sought to detect the
RecBC(HisRecD) complex directly by analyzing mixtures of the proteins
on nondenaturing polyacrylamide gels (32). There was a band present in
the mixture of RecB, RecC, and HisRecD (lane 1, Fig.
3) which co-migrates with RecBCD (lane 3) and
is presumably RecBC(HisRecD). The renatured HisRecD does not form a
clear band (lane 4), indicating that it may not be a single
discrete species. The HisRecD protein would also have little charge and
thus low mobility in the gel (pH 7.4), since its estimated pI is about
6.8, given its content of charged residues. The RecBC(HisRecD) band was
not seen in gels prepared without MgCl2. We believe that
Mg2+ mainly stabilizes the reconstituted enzyme in the gel,
since the nuclease activity of the RecBC(HisRecD) mixture was about the
same whether or not MgCl2 was present during the
reconstitution (not shown).
Reconstitution of Other RecBCD Enzyme Activities
Unwinding ActivityThe rate of DNA unwinding by the RecBCD
enzyme, measured by the RecJ/ExoI coupled assay, is about 4-fold faster
than that of the RecBC enzyme (37). Reconstitution of RecBC with
HisRecD protein gave about a 2.5-fold increase in the rate of DNA
unwinding in this assay (Fig. 4).
Reactions of RecBCD and RecBC(HisRecD)
with a -containing DNA molecule are shown in Fig. 5.
Each enzyme produces a full-length single-stranded product, as well as
DNA fragments which comigrate with the markers from AvaI
digestion. Since the AvaI sites are only 75 nt away from
F, and 13 nt from
H, these fragments serve as markers for
-specific cleavage. The
-specific bands were not produced in
reactions with [5
-32P]pBR322, which lacks
sites, nor
by the RecBC enzyme. This experiment shows that the reconstituted
RecBC(HisRecD) enzyme is able to recognize and cleave double-stranded
DNA near a
sequence.
-Specific cleavage has also been observed
with RecBCD enzyme prepared by reconstituting RecB and RecC with native
RecD protein (32).
ATP Hydrolysis Stimulated by pd(T)12
We found
previously (41) that RecBCD has much greater ATPase activity than does
RecBC in the presence of low concentrations of pd(T)12
oligomers (Km for pd(T)12 = 4.5 µM oligomers for RecBC versus 0.1 µM for RecBCD). We believe that the ATP hydrolysis is
catalyzed by the RecB subunit in both enzymes, since the RecBCD-K177Q mutant enzyme also has a low Km for
pd(T)12, close to that of RecBCD. The reconstituted
RecBC(HisRecD) enzyme also has much greater ATPase activity at 1.5 µM pd(T)12 compared with RecBC alone (Fig.
6). This observation provides further evidence for physical association of the proteins, since we believe that it is the
presence of RecD (wild-type, mutant, or, in this case, HisRecD) in the
RecBCD holoenzyme, that stimulates the activity of RecB. The molecular
basis of this effect is not known.
ATP Hydrolysis by HisRecD Protein
In the course of studying ATP hydrolysis by the reconstituted
enzymes, we also incubated identical samples of RecBC and HisRecD separately, to be used as controls. ATP hydrolysis was then measured at
37 °C. Interestingly, the HisRecD protein alone catalyzed a small
amount of ATP hydrolysis in these experiments with poly(dT) as the DNA
co-substrate (Fig. 7, closed circles), but
the reaction stopped in about 2 min. A second burst of ATP hydrolysis
occurred if more HisRecD was added, but it also stopped in about 2 min (Fig. 7, open circles). Little ATP hydrolysis was observed
if the HisRecD was diluted and placed at 37 °C for 2 min, and then ATP and other reaction components were added to initiate the reaction (Fig. 7, closed squares). There was no detectable ATP
hydrolysis by HisRecD in the absence of DNA (data not shown). These
experiments suggested that HisRecD is a DNA-dependent
ATPase but that it is not very stable and loses activity quickly at
37 °C. Consistent with this conclusion, much greater ATP hydrolysis
by HisRecD was observed when the reaction was done at room temperature
(about 23 °C; Fig. 7, closed triangles). The ATP
hydrolysis activity of HisRecD was not stabilized significantly at
37 °C by the presence of either bovine serum albumin or RecC (not
shown; we did not add RecB, since it is itself an ATPase). The HisRecD
protein must be more stable when it is assembled with RecBC, since the
RecBC(HisRecD) enzyme is very active in the assays shown above, all
carried out at 37 °C, and it is also more stable in long-term
storage than is HisRecD itself.
These experiments provide direct evidence that HisRecD, and presumably
also RecD, is a DNA-dependent ATPase. There is ample reason
to believe that RecD should be an ATPase (see "Introduction"), and
it is unlikely that a contaminant would co-purify with the HisRecD
prepared in this way. We nonetheless carried out the following control
experiment to confirm that the activity is due to HisRecD itself. We
prepared three cultures of JM109 cells, transformed with pHisRecD,
pHisRecD-K177Q, or pTrcHisB (vector-only control). Cell growth,
lysis, and metal-chelate affinity chromatography under denaturing
conditions were carried out as described under "Experimental
Procedures." The fractions eluting from the columns were analyzed by
SDS-polyacrylamide gel electrophoresis. HisRecD was found only in
fractions from pHisRecD or pHisRecD-K177Q-containing cells, and not in
the pTrcHisB control (not shown). All three protein preparations
contained a small amount of an impurity (see above). Fraction number 3 from each preparation was then renatured, and either reconstituted with
RecB and RecC and assayed for nuclease activity, or assayed for ATPase
activity directly. Neither activity was found with the vector-only
control, but addition of either HisRecD or HisRecD-K177Q to RecB and
RecC gave nuclease activity (Fig. 8A). The
nuclease activity was greater with RecBC reconstituted with the
wild-type HisRecD than with the HisRecD-K177Q mutant, as expected based
on previous results (25). However, we observed ATP hydrolysis activity
only with the wild-type HisRecD, and not with HisRecD-K177Q, nor with
the vector-only control (Fig. 8B). This result is consistent
with the conclusion that the observed ATP hydrolysis activity is due to
HisRecD, and also that the Lys to Gln mutation has greatly reduced the
ATPase activity of RecD (as surmised previously but not shown directly
(36, 37)).
Masterson et al. (32) found no ATP hydrolysis activity in
RecD protein purified under denaturing conditions and then renatured. However, RecD would be expected to have little activity in the reaction
conditions apparently used in that work (37 °C, 1.1 nM RecD, 30 µM denatured DNA), if the native RecD protein
behaves similarly to HisRecD. Lieberman and Oishi (20) also did not find ATPase activity with RecD (the " subunit" in that work
(21)), but the protein concentration was unknown, and probably was
quite low.
Characterization of the ATP Hydrolysis Activity of HisRecD and Comparison to RecB
We then studied some of the properties of the HisRecD ATPase activity, and compared it to that of RecB, assayed under identical conditions (these conditions are not optimal for RecB, as that enzyme is somewhat more active at pH 7 than at pH 7.4, and it is active at 37 °C). These experiments were done to obtain comparative information as to the specificity of the two proteins. These properties could of course change when the subunits are in the RecBCD holoenzyme. For example, the ATP hydrolysis activity of the isolated RecB subunit itself is much lower than that of RecB in RecBC or the RecBCD holoenzyme (32, 33, 41).
We initially used high concentrations of poly(dT) to test for ATP
hydrolysis catalyzed by HisRecD, based on the results of previous
experiments with the RecBCD enzyme (41). That enzyme exhibits ATP
hydrolysis activity stimulated by poly(dT) binding at two sites. ATP
hydrolysis by RecB is stimulated by a high affinity site, while DNA
binding in a second site with much lower affinity appeared to stimulate
ATP hydrolysis by RecD. Consistent with those observations, HisRecD has
significant ATPase activity with 1 and 0.1 mM poly(dT)
(average length: 221 nt), but much lower activity with 0.01 mM poly(dT) and almost none with pd(T)12 (Fig. 9). There was also essentially no ATP hydrolysis by
HisRecD with linear double-stranded DNA (XhoI-cut pTZpPB700,
0.01-1 mM nt residues; data not shown). The RecB reaction
rate with 1 mM poly(dT) was about 30-40-fold greater than
that of HisRecD, under the reaction conditions of Fig. 9. The
RecB-catalyzed reaction rate was about the same with
pd(T)12 and poly(dT), each at 1 mM nucleotide
residues (not shown). Thus ATP hydrolysis by RecB was more than
1400-fold faster than that by HisRecD with pd(T)12 as the
DNA co-substrate.
The ATP hydrolysis activities of HisRecD and RecB depend rather
differently on the Mg2+ concentration (Fig.
10). HisRecD was maximal at about 2 mM
Mg2+, and then decreased slightly at still higher
concentrations (Fig. 10A). The activity of RecB increased up
to 10 mM Mg2+, the highest concentration we
tested (Fig. 10B). Thus, the ATP hydrolysis activity of the
two proteins was about the same at low Mg2+ (about 0.5 mM). Both proteins also hydrolyzed Ca·ATP, although less
efficiently than Mg·ATP (not shown). There was no detectable ATP
hydrolysis by HisRecD in the absence of DNA, with 2 mM
Mg2+, under the conditions of Fig. 10 (data not shown). The
Mg2+ concentration has significant effects on the activity
of RecBCD, particularly the relative levels of nuclease and helicase
activity, and the levels of -specific versus nonspecific
cutting (15, 39, 42). We note that RecBCD can unwind DNA when ATP is in excess over the Mg2+ (5, 43), conditions where neither
subunit alone has much ATP hydrolysis activity.
HisRecD hydrolyzes ATP most efficiently, with lower activity with GTP
and UTP, and almost none with CTP (Fig.
11A). ATP is also the best substrate for
RecB under these conditions, but CTP, GTP, and UTP are hydrolyzed at a
rate 35-50% that of ATP (Fig. 11B). The same relative
rates of hydrolysis by RecB were seen at 1 mM NTP, 10 mM MgCl2, whereas we detected hydrolysis solely
of ATP by HisRecD (24 nM) under those conditions (not
shown).
We also tested the HisRecD protein for nuclease activity, using
5-32P-labeled single-stranded oligomers as the DNA
substrate. We have observed no nuclease reaction with either HisRecD
alone, or with HisRecD incubated with either RecC or RecB, at 200 or
500 µM ATP (data not shown). We do detect nuclease
activity with the purified RecBCD enzyme, the reconstituted
RecBC(HisRecD) enzyme, and a low level of activity with the RecBC
enzyme, using this assay.2 These
experiments thus provide no support for the possibility that the RecD
subunit contains the nuclease active site of the RecBCD enzyme, despite
the importance of RecD for this activity (20, 21, 32).
The reconstituted RecBC(HisRecD) enzyme has high levels of all the activities of RecBCD. The levels of activity show that the presence of the 31-residue amino-terminal peptide has little effect on the ability of the fusion protein to reconstitute with the other subunits and to influence the activity of the other subunits. We cannot be certain as to how the peptide might affect the activity and properties of RecD itself. In any case, the availability of this protein and the results we have so far will aid in further study of the function of RecD in RecBCD enzyme activity. HisRecD will also be a useful material for studying the RecD protein by itself, and this subunit reconstituted with the wild-type and with other mutant subunits.
The RecD subunit has been implicated to some degree in all
activities of the RecBCD enzyme (ATP hydrolysis, helicase, nuclease, and the response to sequences). Indeed, it is difficult to assign separate functions to the subunits of the enzyme. Instead it appears that all three participate in the nuclease reaction on single- and
double-stranded DNA, and in unwinding double-stranded DNA. An exception
is that RecB alone has some helicase activity, but it is much slower
than that of RecBCD (6, 44).
The observation of ATP hydrolysis by HisRecD, and its properties, support the conclusion that RecD hydrolyzes ATP during the reaction catalyzed by RecBCD on double-stranded DNA (37). The specificity of HisRecD for single-stranded DNA is consistent with previous conclusions that RecD interacts with the partially unwound DNA produced by the helicase activity driven by ATP hydrolysis by RecB (24, 45, 46). The requirement of HisRecD for high poly(dT) concentrations may result from having isolated the subunit from its partners, since the local concentration of single-stranded DNA at the unwinding fork made by RecBCD would be relatively high, even at low total DNA concentration.
The nucleotide specificities of the two ATPase subunits are interesting in light of previous studies of the RecBCD enzyme. Wright et al. (7) observed double-strand nuclease activity by RecBCD with all eight ribo- and deoxyribonucleoside triphosphates. The activity showed sigmoidal dependence on the nucleotide concentration, and the minimum concentration required for activity went in the order ATP < GTP < UTP < CTP. We found later that the RecBCD-K177Q mutant enzyme required a slightly higher ATP concentration than did RecBCD before double-strand nuclease activity was observed (25), as did RecBC for detectable DNA unwinding (37). We interpreted these observations to indicate that ATP hydrolysis by both RecB and RecD, in RecBCD, contribute to the DNA unwinding and nuclease reactions, at low ATP concentrations. ATP hydrolysis by RecB alone is sufficient for these reactions (in either RecBC or RecBCD-K177Q), but a higher ATP concentration is required. The nucleotide specificity of HisRecD together with the observations of Wright et al. (7) are consistent with this earlier interpretation that ATP hydrolysis by both subunits contributes to the reaction (unwinding and/or nuclease) with double-stranded DNA.
ATP hydrolysis by RecD can have no essential role in RecBCD helicase activity, since RecBC is a helicase (31, 32, 33). The protein may function to accelerate translocation and enhance processivity during unwinding, since RecBC and RecBCD-K177Q are slower and less processive than RecBCD (26, 33, 37). The dependence of ATP hydrolysis rate on DNA length can be diagnostic of a mechanism where ATP hydrolysis is coupled to movement along the DNA (47). Although further study of the kinetics of ATP hydrolysis by HisRecD (or RecD itself) is clearly necessary, our observation that poly(dT) stimulates ATP hydrolysis much more effectively than pd(T)12 is consistent with this proposed function for RecD. Finally, although RecD is dispensable for helicase activity, it cannot be ruled out that ATP hydrolysis by RecD can be coupled to DNA unwinding. A possible mechanism is that RecB must begin to unwind the DNA, but ATP hydrolysis by RecD could also contribute to DNA unwinding once the process has begun.
Finally, RecD is required for the high nuclease activity of RecBCD with
single- and double-stranded DNA (20, 21, 32). Several recent
experiments indicate that inactivates RecD, at least as far as its
function in the nuclease reaction (28-30). The
-inactivated enzyme
does not appear to reactivate readily under some conditions, since a
sequence in one DNA molecule can lead to protection of a
non-
-containing DNA in vitro (9) and in vivo
(12, 29, 30). Overexpression of RecD protein in vivo can
suppress the effect of a
sequence (29, 30), an observation that
supports the proposal that RecD is ejected from the RecBCD holoenzyme
at a
sequence (27, 29, 30), and that the RecD protein is unable to
reassemble when it is present in normal (low) amounts in the cell. Our
results indicate that dissociated RecD subunit would lose activity at
37 °C, if its stability is similar to that of HisRecD. Thus the
results are consistent with the proposal that
sequences act to turn
off RecBCD nuclease activity irreversibly in the cell (30).
We thank Dr. Firouzeh Korangy, Johns Hopkins
University, for assistance with using the pTrcHisB plasmid and working
with the His-tagged protein; Suwei Zhao of the Center for Agricultural Biotechnology, Maryland Biotechnology Institute, for DNA sequencing; and Dr. Gerald Smith and Sue Amundsen for providing the pBR322-FH plasmid and their
-cutting reaction protocol.