(Received for publication, June 5, 1995)
From the
RecBCD enzyme is a multifunctional nuclease that is essential
for the major pathway of homologous genetic recombination in Escherichia coli. It has a potent helicase activity that uses
ATP hydrolysis to unwind very long stretches of DNA. The functional
form of RecBCD enzyme has been unclear, since M of
250,000-655,000 have been previously reported. We have isolated
two oligomeric forms of the enzyme, one (monomeric) containing a single
copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric)
containing two copies of each polypeptide. We show here that the
monomeric form of the enzyme (M
330,000) can
form a stable initiation complex on the end of ds DNA. Depending on the
nature of the ds end, K
estimates ranged
from
0.1 nM to
0.7 nM in the presence of
Mg
ions, which enhanced but was not required for
binding. We further showed that the complex of monomeric RecBCD enzyme
and a ds DNA end was competent to unwind DNA. A general model for the
action of helicases has been proposed that uses repeated conformational
changes between two states of a complex between DNA and a dimeric form
of the enzyme. Our results make such a model unlikely for RecBCD
enzyme.
The RecBCD enzyme (EC 3.1.11.5) is a large ATP-dependent enzyme
that is involved in recombination and repair of DNA in Escherichia
coli (reviewed in (1) ). It is encoded by the recB, recC, and recD genes, whose gene
products have M of 134,000, 129,000, and 67,000,
respectively, as inferred from DNA sequence data and N-terminal peptide
analysis(2, 3, 4, 5) . The enzyme
has a potent ATP-dependent exonuclease that is active on either ds (
)or ss DNA and a weak ATP-stimulated endonuclease activity
that acts only on ss DNA. It can use the energy of ATP hydrolysis to
unwind ds DNA, either transiently or permanently, in a highly
processive reaction(6, 7) . The enzyme is active on
linear, but not circular, ds DNA and thus requires a ds terminus for
its unwinding or nuclease activity(8, 9) .
Published reports suggest that RecBCD enzyme can exist in either a
monomeric (BC
D
) or a dimeric
(B
C
D
) form. (
)The native M
of the enzyme was initially reported to be about
250,000(9) , as estimated by glycerol gradient centrifugation,
consistent with the enzyme molecule containing one copy each of the
RecB, RecC, and RecD polypeptides (with a predicted M
of 330,000). A higher M
form of the enzyme,
apparently dimeric, was observed in sonicates of E.
coli(10) , together with the previously reported form, but
was lost during subsequent purification steps. RecBCD enzyme purified
from a strain that overproduced the enzyme had a native M
of 655,000 but which decreased to about 270,000
in the presence of 0.5 M NH
Cl(11) . The
higher M
form was not observed in a subsequent
purification(12) . More recently, RecBCD enzyme has been
produced either by overproduction of the three subunits within E.
coli or by mixing of purified subunits(5) . M
estimations by gel filtration or native
polyacrylamide gel electrophoresis are consistent with the enzyme from
these sources being a monomer, but the specific activity of the
reconstituted material was only a few percent of that of the native
enzyme(5) .
Interest in the subunit structure of RecBCD enzyme was rekindled by a ``rolling dimer'' model of helicase action which relies on a symmetric, dimeric enzyme structure(13) . In that model, monomer A of the dimeric helicase binds (already unwound) ss DNA behind the enzyme, and monomer B binds ds DNA immediately ahead. Monomer A releases its ss DNA and binds to ds DNA ahead of monomer B, which melts its bound ds DNA and remains bound to the ss DNA so produced. Translocation and unwinding thus result from the cycle of alternating binding and unbinding.
Unwinding of ds DNA by RecBCD enzyme has been studied by electron
microscopy(6, 14) . The enzyme unwinds DNA
processively, in the presence of SSB, with the production of either
asymmetric structures (a ss loop and two ss tails) or apparently
symmetric structures (two ss loops). Both types of structure travel
along the DNA at 300 bp/s, while the loops grow at about 100 nt/s.
The relative abundance of the two structures is determined by the
concentration of SSB(15) , suggesting that they arise by a
common mechanism(6) .
We proposed that, as a minimal model, RecBCD enzyme need contact only one strand of ds DNA and could produce the observed DNA structures by assimilating the DNA ahead of itself and releasing it behind itself at a slower rate(6) . Roman and Kowalczykowski (16) proposed that unwinding by RecBCD enzyme results from the action of two helicases, acting at different rates on the two strands of the DNA, and suggested that the helicases may reside in the RecB and RecD subunits of the enzyme. Ganesan and Smith (17) combined the latter model with the ``rolling dimer'' model of Lohman (13) and suggested that a dimeric form of RecBCD enzyme unwinds DNA. Two copies of the RecB subunit were proposed to act in tandem to translocate along one strand of the DNA, while a pair of (RecC + RecD) complexes translocated along the other strand. The two complexes were postulated to travel at different rates and hence produce the ss loops observed by electron microscopy. The choice of subunits in this latter model was prompted by the UV-cross-linking patterns of the subunits on the ends of ds DNA(17) , by the abilities of the RecB and RecD subunits to bind ATP(18) , and by the ATPase activity of isolated RecB protein(19) .
During the course of purifying RecBCD enzyme we observed, purified, and characterized both monomeric and dimeric forms of RecBCD enzyme. We report here that stable complexes were formed between ds DNA and the monomeric form of RecBCD enzyme and that such complexes were competent for unwinding. These results establish the active form of RecBCD enzyme as a monomer and question the physiological relevance of the dimer.
In the ``second'' purification, Fraction V was purified as previously described (8, 21) and concentrated and purified on a DEAE-Sepharose column, as above, to yield Fraction VII, which lacks detectable contaminants as judged by SDS-polyacrylamide gel electrophoresis.
Units of activity are those of the ATP-dependent ds
DNA exonuclease activity of the enzyme(22) . RecBCD enzyme
concentrations were derived from the A of enzyme
fractions, using the
calculated by Roman and
Kowalczykowski (7) .
DNA concentrations
are given as molarities of DNA molecules, unless otherwise stated, and
were calculated from the A of the plasmid DNA
solutions and the specific activities of the labeled DNA species, as
measured by trichloroacetic acid precipitation(22) .
Figure 1: Glycerol gradient separation of two forms of RecBCD enzyme. Sedimentation, collection, and assays were as described under ``Experimental Procedures.'' Sedimentation was from right to left. A, purification of Fraction V-B. B and C, recentrifugation of purified monomer and dimer fractions, respectively, from a previous glycerol gradient purification of part of Fraction V-B. The protein concentration, ds DNA exonuclease activity, and specific activity of each glycerol gradient fraction are expressed as a fraction of the maximum observed in that panel.
Material equivalent to Fractions VI-M and VI-D, obtained from a previous glycerol gradient purification, was analyzed in the same experiment. The monomeric fraction from that gradient contained very little of the ``dimer'' species (Fig. 1B), while the ``dimer'' material contained approximately equal weights of the two forms of the enzyme (Fig. 1C). The origin of the monomer-sized material in this ``dimer'' fraction is unclear: it may have arisen from inefficient purification in the previous glycerol gradient, or it may have resulted from instability of the dimer form of the enzyme. Observations with nondenaturing gels (see below) suggest that the dimeric material also returns to the monomeric state during electrophoresis, suggesting that the dimer may indeed be unstable.
To estimate the relative numbers of copies of each polypeptide in
the two forms of RecBCD enzyme, fractions VI-M and VI-D were analyzed
by SDS-polyacrylamide gel electrophoresis, and the Coomassie-stained
gels were quantitated by densitometry. The observed relative
intensities for the three polypeptides were within 15% of those
predicted for a protein with one subunit each of 134,000, 129,000, and
67,000 (Table 2), as previously reported (7, 29) for purified enzyme. While the relative
Coomassie staining abilities of the RecB, RecC, and RecD polypeptides
have not been measured, the relative staining abilities of several
proteins are proportional to the number of positively charged amino
acids they contain(30) . As shown in Table 2, the three
polypeptides of RecBCD enzyme contain similar densities of positively
charged amino acids. Hence, the equimolar staining of the three
polypeptide bands is consistent with RecBCD enzyme containing equal
numbers of copies of the RecB, RecC, and RecD polypeptides. This
result, together with the estimated M of monomeric
enzyme, shows that monomeric enzyme contains one copy of each
polypeptide.
Quantitation of a Coomassie-stained native polyacrylamide gel showed that >60% of the protein in Fraction VI-D was in the dimeric form and that 95% of Fraction VI-M was in the monomeric form. Hence, the observation of equal ratios of polypeptides in the two forms (Table 2) implies that the dimeric form in Fraction VI-D also contained equal numbers of each subunit. The faster sedimenting form of RecBCD enzyme must thus be a simple dimer of the monomeric form, with two copies each of the RecB, RecC, and RecD polypeptides.
Fig. 1also shows the ds DNA exonuclease activity and specific activity of each gradient fraction. The specific activity of the dimeric enzyme was less than 25% of that of the monomeric enzyme. Exonuclease activity of the dimeric enzyme was apparently not due to contamination with monomeric enzyme, as the specific activity of the dimeric enzyme was constant across the peak of dimer material (Fig. 1, A and C). One explanation for a lower specific activity, a dimer with the active site of one monomer occluded, would predict a specific activity half that of the monomer, greater than that observed. Alternatively, the dimeric form may be devoid of ds DNA exonuclease activity and the observed nuclease activity may be that of monomeric enzyme that has arisen from the dimer before or during the assay.
The M of the monomer
form estimated by this method is consistent with those measured by
glycerol gradient centrifugation (preceding section) and by native gel
electrophoresis (next section). These results confirm that the major
form of the enzyme is indeed a monomer.
Figure 2:
Molecular weight estimates and composition
of RecBCD enzyme fractions determined by nondenaturing polyacrylamide
gel electrophoresis. A high pH nondenaturing discontinuous 5-20%
gradient gel (31) was run for 2 h in the cold room; the current
was adjusted to maintain the upper buffer chamber temperature below 10
°C. Lane 1 contained 0.5 µg each of thyroglobulin,
ferritin, catalase, bovine serum albumin (M 67,000
and 134,000), and ovalbumin. The other lanes contain fractions from the
first RecBCD enzyme purification. Lane 2, Fraction VI-M (790
units, 2.2 µg); lane 3, Fraction VI-D (1 µg); lane
4, Fraction IV (1700 units, 11 µg). Proteins were detected by
Zoion Fast Stain. Linear regression of log(M
) versus migration distance for the 669,000, 440,000, and
232,000 markers (r = -0.999) was used to estimate
the M
s of monomer (310,000) and dimer (630,000) in lanes 2 and 3.
Figure 3:
Nondenaturing polyacrylamide gel
electrophoresis of RecBCD enzyme from two enzyme preparations. A 5%
polyacrylamide gel in 50 mM MOPS-KOH (pH 7.0), 2 mM EDTA was run for 2 h at 100 V. Lane 1 contained 1 µg
of bovine serum albumin, providing M markers of
67,000, 134,000, and 200,000. Lane 15 contained 2 µg each
of thyroglobulin (669,000) and ferritin (440,000). Lanes 2, 5, 7, 9, and 11 contained Fractions
VI-D (0.7 µg) from the first purification. Lane 3 contained Fraction IV (500 units, 3.3 µg), and lane 4 contained Fraction VI-M (550 units, 1.5 µg), both from the
first purification. The remaining lanes contained fractions from the
second purification. Lane 6, Fraction VII (490 units, 4.1
µg); lane 8, Fraction V (110 units, 0.6 µg); lane
10, Fraction IV (85 units, 1.4 µg); lane 12, Fraction
IV (170 units, 2.8 µg).
In a nondenaturing
4-15% polyacrylamide gradient gel, at pH 7.5, the protein
standards migrated as a smooth function of their Ms (data not shown). The apparent M
of the monomer was 230,000, and that of the dimer was about
670,000, suggesting that RecBCD enzyme may be more highly charged than
the standards at pH 7.5. The isoelectric points of the proteins, as
calculated from their primary sequences using the
``Isoelectric'' program of the GCG Software
package(32) , were consistent with this interpretation. RecBCD
enzyme had a calculated pI of 5.0, while those of the protein standards
were higher: thyroglobulin, 5.4; ferritin, 5.5; catalase, 6.9; and
bovine serum albumin, 6.0.
For many experiments, 5% polyacrylamide
gels were used and were run at the pH (7.0) used in the DNA binding and
unwinding experiments reported below. Such gels easily separated the
monomeric and dimeric forms of RecBCD enzyme, as shown in Fig. 3, although the apparent Ms of the
enzyme fractions, relative to the protein standards, were considerably
lower (about 600,000 for the dimer and about 150,000 for the monomer),
again probably due to the higher relative charge, at pH 7.0, on RecBCD
enzyme than on the standards.
A significant fraction of the protein migrated faster than RecBCD enzyme in these gels (Fig. 3, lanes 3, 6, 10, and 12). This material may have arisen from enzyme molecules lacking the RecD subunit, as has been observed by others(5) . It was not detectable in the glycerol gradient purified enzyme (Fraction VI-M; Fig. 3, lane 4), showing that it was a separate species and not the result of instability of the enzyme during gel electrophoresis. The proportion of dimeric RecBCD enzyme in Fraction VI-D appears higher in Fig. 3(lane 2) than in Fig. 2(lane 3), probably due to the instability of the enzyme at the higher pH in the gel in Fig. 2. All purified dimer fractions prepared by glycerol gradient centrifugation appeared, on analysis in gels similar to that shown in Fig. 3, to contain similar amounts of monomeric RecBCD enzyme. It is thus plausible that the different samples of dimeric material contained principally dimeric RecBCD enzyme, but that the dimeric enzyme was unstable both during glycerol gradient centrifugation (Fig. 1) and during gel electrophoresis (Fig. 3).
Origin of the Dimeric Form-Examination of the different stages of purification of RecBCD enzyme suggests that at least some of the dimeric species arose during purification ( Fig. 2and Fig. 3). In Fig. 2, lane 4, it appears that Fraction IV of the first purification contained no detectable RecBCD enzyme dimer. However, when this fraction was subsequently reanalyzed on the pH 7 gel system (in which RecBCD enzyme is more stable), some material was visible that comigrated with the dimer (Fig. 3, lane 3). It is unclear which gel is a more accurate reflection of the true makeup of Fraction IV. Dimer might not have been detected in Fig. 2, due to its instability in that gel system. The band comigrating with dimer in Fig. 3might be a contaminant rather than RecBCD enzyme or it might be dimeric RecBCD enzyme produced on storage of Fraction IV. Nonetheless, it is clear that, while there may have been a small amount of dimer present in Fraction IV of the first purification (Fig. 3, lane 3), the proportion present in Fraction V-B is considerably greater (Fig. 1A).
In the second purification, protein comigrating with the dimer was barely detectable in Fraction IV (Fig. 3, lanes 10 and 12). Dimer was not detectable in Fraction V (lane 8), perhaps due to the small amount of material that could be loaded on the gel, but was detected when a gel similar to that in Fig. 3was silver-stained (data not shown). In Fraction VII (lane 6), dimer was clearly present at a higher concentration, relative to the monomeric form of RecBCD enzyme, than it was in Fraction IV. Dimer was thus apparently generated during the late stages of both purifications. It seems plausible that the greater concentrations of RecBCD enzyme achieved in the latter stages of purification may be responsible for production of the dimer.
Figure 4:
End-specific binding by RecBCD enzyme
monomers. DNA species were incubated with the indicated concentrations
of RecBCD enzyme (Fraction VI-M), in 10 µl of 20 mM MOPS-KOH (pH 7.0), 4 mM Mg(OAc), 100
µg/ml polyvinylpyrrolidonone K-60, for 10 min at 20 °C, before
addition of 2.5 µl of 50% glycerol and analysis in a 5%
polyacrylamide gel in 20 mM MOPS-KOH (pH 7.0), 4 mM Mg(OAc)
. Samples in lanes 1-5 contained
0.05 nM dimerized 5`-
P-labeled hairpin-shaped BamHI oligonucleotide, while those in lanes 8-12 contained 0.1 nM 5`-
P-labeled hairpin-shaped
oligonucleotide. The samples in lanes 13-15 contained 1
nM nicked hairpin DNA and were aliquots (taken immediately
prior to the addition of ATP plus heparin) of the reaction mixtures
used in lanes 3-5 of Fig. 5. Lanes 6 and 7 contained Fractions VI-D and VI-M, respectively, of RecBCD
enzyme, which were detected by Coomassie staining of the gel. The
corresponding portion of the stained gel is aligned over lanes 6 and 7 of the autoradiograph.
Figure 5:
Unwinding of DNA by RecBCD enzyme
monomers. RecBCD enzyme was incubated with the 170-bp P-labeled nicked hairpin DNA (1 nM) for 10 min at
20 °C to allow binding, samples taken from lanes 3-5 for analysis in the gel in Fig. 4, and RecBCD enzyme
reaction started by addition of ATP (mixed, in the lanes indicated,
with reaction inhibitors). In lanes 2, 6, and 8, the inhibitors were added to the reaction mixture prior to
RecBCD enzyme, and the ``reaction'' was started by the
addition of ATP. The faint band migrating between the positions of ds
and unwound hairpin-capped DNA is a single strand of uncapped DNA, and
arises from unwinding of uncapped DNA that contaminated the substrate.
Capped and uncapped ds DNAs were not separated in this gel.
ds DNA molecules with two ends can bind a RecBCD enzyme molecule at each end and are retarded to the position of dimeric RecBCD enzyme (data not shown). Thus, DNA bound at one end by dimeric RecBCD enzyme, or by two monomers, cannot be distinguished from DNA molecules with monomeric RecBCD enzyme molecules bound at each end.
To test whether the initiation complex of RecBCD enzyme on a ds DNA end involved more than one RecBCD enzyme monomer, we used a ds DNA substrate with one terminus masked by a ss loop, called a ``hairpin,'' as shown in Fig. 4. A dimer of that substrate, with no strand ends, was used to test whether binding was end specific. ds DNA with such ss loops at its termini is resistant to digestion or cleavage by RecBCD enzyme(27, 33) . About half of the hairpin DNA with one ds end (1 nM total concentration) was retarded by 0.3 nM monomeric RecBCD enzyme (lane 10), and essentially all of it was retarded by 1 nM RecBCD enzyme (lane 9). Very little of the endless DNA was retarded at any RecBCD enzyme concentration (lanes 2-5), showing that the binding of RecBCD enzyme was indeed to the ends of DNA molecules. The great majority of the retarded DNA molecules were retarded to the position of free monomeric RecBCD enzyme. A monomer of RecBCD enzyme is thus sufficient to form a complex at the end of a ds DNA molecule.
At the highest RecBCD enzyme concentrations (1 and 3 nM), a small fraction of the endless hairpin DNA was retarded to the position of RecBCD enzyme monomers (lanes 4 and 5), presumably from end-independent binding by RecBCD enzyme. At 3 nM RecBCD enzyme, 15% of the retarded hairpin DNA (with one ds end) was retarded to the position of dimeric RecBCD enzyme, as measured by PhosphorImager analysis of the gel in Fig. 4(lane 8). As 95% of the RecBCD enzyme in Fraction VI-M was monomeric (see above), this retardation to the dimer position presumably resulted from one enzyme molecule binding to the end of hairpin DNA and a second molecule binding elsewhere.
We next investigated the effects of Mg ions and the nature of the ds DNA end on the ability of RecBCD
enzyme to bind DNA. Hairpin DNA with 5`-overhangs of 0-4 nt were
mixed with RecBCD enzyme and analyzed by gel retardation in the
presence or absence of Mg
ions. The DNA concentration
was kept constant and the protein concentration was varied. Analysis of
Hill plots showed that the K
for DNA with a 4-nt
5`-overhang was 0.56 nM (Table 4), in reasonable
agreement with the data in Table 3in which the DNA concentration
was varied. Since the estimates of K
in Table 3and Table 4depend on knowing the absolute DNA and
RecBCD enzyme concentrations, respectively, uncertainties of these
values contribute to the differences in these estimates. Binding was
tighter to DNA with a 4-nt 5`-overhang (K
=
0.08 nM) in the presence of Mg
ions. As the
length of the 5` extension was decreased from 4 to 0 nt, the K
increased, about 7-fold in the presence of
Mg
ions and about 10-fold in its absence. In each
case binding was tighter in the presence of Mg
ions
than in its absence. For blunt-ended DNA (0-nt overhang) in the absence
of Mg
ions the K
was 6.6
nM, again in reasonable agreement with the data in Table 3in which the DNA concentration was varied. Even higher K
values (50-250 nM) were measured
with an 80-nt ss DNA substrate in the absence of Mg
ions (data not shown). Thus, ds DNA with a short ss overhang
appeared to be the preferred substrate for RecBCD enzyme binding, at
least in the absence of Mg
ions; Mg
ions enhanced, but were not required for, binding.
In the
experiments in Fig. 5, RecBCD enzyme was allowed to bind to the
end of DNA, in the presence of Mg but the absence of
ATP, and the reaction started by the addition of a mixture of ATP and
one of the inhibitory agents. The DNA substrate was a 170-bp ds DNA,
blocked at one end with a hairpin oligonucleotide. The substrate had a
nick adjacent to the hairpin, to allow separation of the strands by
RecBCD enzyme without the need for nuclease activity.
Heparin(34) , Sarkosyl(35) , or excess unlabeled ds DNA
molecules were used to prevent reinitiation by RecBCD enzyme. Each of
the agents was effective at preventing unwinding: addition of the agent
prior to RecBCD enzyme prevented detectable unwinding when ATP was
subsequently added (lanes 2, 6, and 8). When
added after the enzyme, unlabeled DNA had little effect on the extent
of unwinding (lanes 7 versus 10), while Sarkosyl decreased
unwinding slightly (lanes 9 versus 10). Heparin inhibited
unwinding by about 50%; at 2 nM RecBCD enzyme about half as
much unwound DNA was produced in the presence of heparin as in its
absence (lanes 4 versus 10), but the inhibition was overcome
by doubling the RecBCD enzyme concentration (lanes 5 versus
10).
Complexes were formed between 1 nM nicked (170
bp) hairpin DNA molecules and 1, 2, or 4 nM RecBCD enzyme, and
a portion of each was analyzed on a nondenaturing gel (Fig. 4, lanes 13-15). With 4 nM RecBCD enzyme, all of
the DNA was retarded, while with 2 nM enzyme, approximately
half of it was. The retarded DNA migrated more slowly than monomeric
RecBCD enzyme but faster than the dimeric form. The 35-bp
DNARecBCD enzyme complex comigrated with unbound monomeric enzyme (Fig. 4, lane 8), while the 70-bp DNA
RecBCD
enzyme complex was slightly retarded (lane 5) and the 170-bp
DNA
RecBCD enzyme complex more retarded (lanes 14 and 15). The retardation was thus length-dependent, and all of the
complexes contained one monomeric RecBCD enzyme molecule per DNA.
The remainder of the complexes were allowed to react, by addition of ATP and the blocking agents, and unwinding monitored by gel assay (Fig. 5). With 2 nM RecBCD enzyme half of the DNA was retarded (and hence in a complex; Fig. 4, lane 14), and approximately half of it was unwound (Fig. 5, lane 7) when unlabeled DNA was used to prevent reinitiation by RecBCD enzyme. Hence, nearly all of the complexes appear competent for unwinding.
With 4 nM RecBCD enzyme, all of the DNA was bound to RecBCD enzyme (Fig. 4, lane 15), but only about half of it was unwound when reaction was initiated by addition of ATP plus heparin (lane 5). However, as noted above, heparin appeared to inhibit unwinding by about 50%, and so it would again appear that all the complexes were competent to unwind.
In summary, the initiation complex between RecBCD enzyme and a ds DNA end contained one RecBCD enzyme monomer per DNA end, and virtually every such complex appeared to be competent to unwind DNA.
We (Fig. 1) and others (10, 11) observed a
second form of RecBCD enzyme with higher M. This
form has the same molar ratio of the three polypeptides (1:1:1) as the
monomer (Table 2). Its M
was estimated by
sedimentation rate to be about 600,000 (Table 1) or 655,000 (11) or by native gel electrophoresis at high pH to be 650,000
( Fig. 2and ``Results''). Thus, this form appears to
be a simple dimer (B
C
D
) of the
monomer. The form of RecBCD enzyme observed in cell lysates (10) may arise from the binding of one monomer to each end of
ds DNA fragments generated during lysis (see below). The dimer observed
in purified enzyme may stem from the high concentration achieved during
purification (Fig. 3). These considerations, plus the low
specific activity of the dimeric form (Fig. 1), make us doubt
that the dimeric form is physiologically relevant.
Binding of ds DNA to RecBCD enzyme, as measured
by filter binding, is dependent on Mg ions (17) but the binding measured by gel retardation reported here
occurred without Mg
ions. Presumably Mg
ions are required for the binding of the protein to the filter,
rather than for the binding of the DNA to the protein. The Hemophilus influenzae homolog of RecBCD enzyme does not
require Mg
ions for filter binding and has a K
, as measured by the ratio of dissociation to
association rate constants, of 0.5 pM(36) , about
100-fold lower than that reported here. The K
for
the binding of RecBCD enzyme to flush-ended ds DNA has been measured,
by filter-binding assays, to be 7 pM(29) . The reason
for the 250-fold difference between the values obtained by
filter-binding and by gel-shift assays (Table 3, last line) is
unknown. Wilcox and Smith (35) also concluded, for the Hemophilus enzyme, that the complex contained one enzyme
molecule per DNA end, based on the noncooperative nature of the
binding, but were unable to determine unambiguously whether the active
form of the enzyme was a monomer or a dimer.
One ds DNA break in E. coli would produce about 4 nM ds DNA
ends(37) . Essentially all such breaks would thus be bound by
the available RecBCD enzyme, as the enzyme concentration (100
nM) and the concentration of ends are both significantly above
all estimates of the K
. RecBCD enzyme is thus
likely to attack efficiently any ds DNA end introduced into the cell.
We thus conclude that a monomer of RecBCD enzyme can bind to the end of ds DNA and translocate along and unwind the DNA. The simple form of the dimeric unwinding model for RecBCD enzyme (17) based on that of Lohman (13) thus cannot be correct. As RecBCD enzyme is a large multisubunit enzyme, one might consider combinations of existing helicase models. The RecBCD model of Roman and Kowalczykowski (16) utilizes two helicases, one acting on each strand, and the helicase model of Lohman (13) requires two (quasi) equivalent binding sites for each helicase. Hence, for a monomer of RecBCD enzyme to unwind DNA by such a mechanism, two sets of quasi-equivalent binding sites would be required. However, only one putative ATP binding site has been identified in the RecB and RecD subunits, and there is no evidence for repeated structures in any of the enzyme subunits(2, 3, 4) . It thus seems unlikely that RecBCD enzyme unwinds DNA via the Lohman (13) dimer model.
Knowledge of the minimal complex necessary for unwinding to occur should facilitate determining the mechanism by which RecBCD enzyme unwinds DNA. Electron microscopy of RecBCD enzyme and further characterization of its complex with a ds DNA end may yield important clues in this quest.