(Received for publication, May 4, 1995)
From the
The T4 bacteriophage Dda helicase is believed to be involved in
early events in T4 DNA replication and has been shown to stimulate
genetic recombination processes in vitro. Dda unwinds
double-stranded DNA with 5` to 3` polarity but its ability to
translocate on DNA has not been established. The DNA stimulated ATPase
activity of Dda helicase has been used to probe translocation on
single-strand DNA (ssDNA). Dda exhibits higher ATPase activity in the
presence of poly(dT) than oligo(dT), indicating that Dda
translocates on ssDNA. Oligonucleotides containing biotin/streptavidin
blocks on the 5` or 3` end were used to probe directionality of
translocation. The K
(K
for DNA) for Dda ATPase activity was reduced in the presence
of a streptavidin block on the 3` end, whereas a streptavidin block on
the 5` end had only a small effect on the steady-state ATPase
parameters. These results suggest that Dda translocates
unidirectionally in a 5` to 3` manner and upon encountering the block
remains bound to the oligonucleotide rather than sliding off the 3`
end. The direction of translocation on ssDNA is consistent with the
direction in which Dda unwinds duplex DNA and is not dependent on
duplex structure.
Double-stranded (ds) ()DNA must be melted to provide
single strands (ss) for processes such as replication, recombination,
and repair. The helicase enzymes responsible for providing ssDNA
(Matson and Kaiser-Rogers, 1990; Lohman, 1993) apparently utilize
energy available from nucleotide triphosphate hydrolysis to disrupt the
molecular interactions which stabilize dsDNA. However, the mechanism(s)
by which the helicases transduce chemical energy to mechanical work
necessary for dsDNA melting has not been established. Helicase binding
to ssDNA, or in some cases dsDNA, stimulates their nucleotide
triphosphatase activity, which in turn may drive protein conformational
changes required for function (Wong and Lohman, 1992; Chao and Lohman,
1990; Geiselmann et al., 1993).
The mechanism of dsDNA unwinding by the helicase may be related to its mechanism of translocation on ssDNA. Helicases have been categorized by their unwinding directionality, namely whether they unwind in a 5` to 3` or 3` to 5` fashion, relative to the strand on which they bind. This directional preference observed for dsDNA unwinding implies that helicases may translocate unidirectionally on ssDNA. Although translocation by the enzyme along ssDNA has been shown for a number of helicases, unidirectional translocation on ssDNA has generally not been demonstrated (Young et al., 1994b).
Lohman (Wong and Lohman, 1992) has proposed a rolling mechanism for the Escherichia coli Rep helicase mode of action. This enzyme functions as a dimer in which each monomer provides a DNA binding site. Binding of one monomer to ssDNA frees the second to bind and unwind dsDNA at a ss/dsDNA junction. The proposed mechanism for E. coli Rep helicase does not necessarily require unidirectional translocation on ssDNA but relies on the polarity of the ss/dsDNA junction to provide directionality in dsDNA unwinding. von Hippel and co-workers (Geiselmann et al., 1993) have proposed a model for E. coli Rho helicase in which the enzyme translocates with a 5` to 3` directional bias and ``unzips'' an RNA/DNA hybrid. This mechanism does imply unidirectional translocation by the enzyme on ssRNA. Finally, it is possible that a helicase might unwind dsDNA without translocating via a ``stoichiometric'' mechanism. In this case, helicase binding near a ss/dsDNA junction induces formation of a region of ssDNA distal from the helicase binding site, to which a second molecule of the helicase may bind. The ss/dsDNA junction may thus be propagated unidirectionally without actual helicase translocation.
The bacteriophage helicase Dda (DNA-dependent ATPase) has been implicated in early events in T4 DNA replication, although its precise role has yet to be determined (Barry and Alberts, 1994; Gauss et al., 1994). Dda has also been shown to stimulate the rate of DNA branch migration catalyzed by the T4 UvsX protein, a recombinase similar in function to E. coli Rec A protein (Kodadek and Alberts, 1987). Dda helicase unwinds DNA in a 5` to 3` direction in a distributive fashion (Jongeneel et al., 1984) with scant evidence for its translocation on DNA. In this report, the ATPase activity of Dda on different DNA strands has been used as a means to probe the possible translocation of the enzyme along ssDNA. We have constructed DNA strands of varying length as well as DNA strands containing potential blocks to translocation in order to investigate whether this enzyme translocates on ssDNA and, if so, whether translocation is directionally biased.
Figure 1: Sequences of oligonucleotide substrates for unwinding studies.
The 60-mer fork and 5` tail substrates were unwound to a similar degree under these conditions. However, the 3` tail and 30-mer duplex substrates were not measurably denatured during the 10-s reaction time. Thus, Dda unwinds DNA with a 5` to 3` polarity as previously shown but does not unwind a blunt end duplex oligonucleotide. The fact that the unwinding is directional suggests that the helicase may translocate in a 5` to 3` direction on ssDNA. This presumption is not necessarily valid (Young et al., 1994b). Translocation on ssDNA might not be directionally biased, if unwinding is unidirectional only as a consequence of the ss/dsDNA junction inducing the directionality (Lohman, 1993). Since Dda unwinds dsDNA in a distributive fashion, rapidly dissociating from DNA during the progress of the reaction, this helicase may not translocate at all (Raney et al., 1994; Jongeneel et al., 1984; Krell et al., 1979). Direct evidence for Dda translocation on ssDNA is required.
Figure 2:
Binding of Dda helicase with poly(dA).
Binding of Dda to poly(dA) was investigated by fluorescence quenching
and by ATPase activity. A, ATPase activity was measured as a
function of poly(dA) using the coupled ATPase assay described under
``Experimental Procedures.'' Dda concentration was 50
nM, and KOAc concentration was 160 mM. The K of 31 ± 1.8 µM nt was
obtained by fitting the data to the Michaelis-Menten equation. B, fluorescence titration of Dda (150 nM) with
poly(dA) in 25 mM Tris-OAc, pH 7.5, 160 mM KOAc at 25
°C. The data were fit to the quadratic equation, yielding a K
of 8 ± 1.0 µM nt.
A simple kinetic model (Fig. S1) describing steady-state ATPase activity for ATP-driven
translocases has been proposed (Young et al., 1994a). The
translocation process is represented by k, which
is the rate constant for dissociation of the enzyme from the end of the
DNA lattice via translocation and is dependent on DNA length. All
enzyme-DNA species which are capable of hydrolyzing ATP are combined
into one term, E
DNA, which hydrolyzes ATP with the rate
constant k. The terms describing V
(, e is the total enzyme concentration) and K
(the K
for DNA, ) for this simple model predict that increasing DNA length
should result in decreased values of K
while not
changing V
. This model provides a useful context
in which to analyze DNA dependent ATPase
activity.
Figure S1: Scheme I.
Figure 3: ATPase activity on varying length oligonucleotides. Sequences of oligonucleotides used to investigate the dependence of Dda ATPase activity on DNA length.
Figure 4:
Dda
ATPase activity is dependent on substrate length. ATPase activity was
measured in the presence of varying concentrations of poly(dT)
() and oligo(dT)
(
) using a Dda concentration
of 25 nM. Data were fit to the Michaelis-Menten equation (solid lines). The V
and K
values for poly(dT) were 2660
nM
s
and 300 nM nt, whereas V
and K
for
oligo(dT)
were 1920 nM
s
and 690 nM nt, respectively.
Figure S2: Scheme II.
This
mechanism is similar to that outlined by Young et al. (1994a),
with the difference being that the enzyme species
[EDNA]" in Fig. S2can hydrolyze ATP,
whereas a similar species described by Young could not hydrolyze ATP.
The resulting terms describing V
and K
are shown in and ,
respectively.
The rate constant which is dependent on DNA length, k, now appears in both the V
and K
terms, indicating that both kinetic
parameters can be affected by DNA lattice length, as seen in Fig. 4.
The ATPase
activity of Dda was measured using a DNA fork substrate to determine if
any difference existed in stimulation of ATPase activity during
unwinding of dsDNA as compared with binding to ssDNA, Fig. 5.
Previous work (Raney et al., 1994) had shown that Dda unwinds
the 60-mer fork until the unwinding rate and reannealing rate of
product reach a steady state (50% when Dda is 50 nM and
the 60-mer fork is 250 nM). Thus, the ATPase activity measured
under steady-state conditions represents Dda ATPase activity that is
stimulated partially by unwinding of the 60-mer fork DNA and partially
by binding to ss 60-mer. ATPase activity is stimulated very little by
blunt end dsDNA, since a 30-mer duplex at 250 nM gave rise to
ATPase activity of 154 nM
s
, whereas
the ssDNA 60-mer at the same concentration produced a rate of 3400
nM
s
(data not shown). The ATPase data
in Fig. 5is plotted in terms of the concentration of 60-mer
strands in order to compare directly the quantity of DNA used in each
experiment (see Fig. 1for sequences of the 60-mer and the
60-mer fork substrates). The data provided identical K
values for each substrate of 150 nM strands, whereas V
for the ssDNA 60-mer was slightly higher than
the 60-mer fork, 4940 nM
s
versus 3890 nM
s
. No conclusion can be
drawn with regard to translocation directionality of the Dda helicase,
except that the enzyme's ATPase activity is insensitive to the
single strand or duplex nature of the DNA fork substrate.
Figure 5:
Dda ATPase activity on single strand
60-mer and partial duplex 60-mer fork substrates. ATPase activity was
measured as a function of concentration of ssDNA 60-mer strands
() and a partially duplex 60 mer fork molecules (
). For
comparison, results are shown in terms of concentration of 60-mer
strands, i.e. one 60-mer fork contains two 60-mer strands. Dda
concentration was 50 nM. The V
and K
for the 60-mer ssDNA were 4940
nM
s
and 150 nM strands,
whereas V
and K
for the
60-mer fork were 3890 nM
s
and 150
nM strands, respectively.
Finally,
T4 32 single strand-binding protein was bound to ssDNA in order to
impede either binding and/or translocation of the Dda helicase. Data
from the inhibition of T4 41 helicase ATPase activity by T4 32 single
strand-binding protein has been successfully analyzed in terms of such
a model (Young et al., 1994b). Dda ATPase activity was
measured using poly(dT) and varying concentrations of 32 with the
results being as shown in Fig. 6. At low 32 concentration, a
small but reproducible enhancement of Dda ATPase activity occurs.
Inhibition ensues at higher levels of 32, but the degree of inhibition
does not follow that expected if 32 is preventing Dda from binding. The
concentration of 32 necessary to completely cover the DNA is 71 nM based on a 7-nucleotide site size for 32 (Kowalczykowski et
al., 1981). Inhibition at this concentration is only 30%
which agrees qualitatively with previous reports (Jongeneel et
al., 1984). The degree of inhibition is insufficient to pursue the
analysis described by Young et al. (1994b).
Figure 6: Inhibition of Dda ATPase activity by the T4 gene 32 single strand-binding protein. ATPase activity was measured with 25 nM Dda, 500 nM poly(dT), and varying amounts of 32 protein. The concentration of 32 protein necessary to completely cover the DNA is 71 nM, assuming a site size of 7 nt for 32 protein (Kowalczykowski et al., 1981). Dda ATPase activity is only partially inhibited, indicating that Dda is able to compete with 32 for DNA under these conditions.
Figure 7:
Effect of biotin/streptavidin blocks on
Dda ATPase activity. ATPase activity of 25 nM Dda was measured
in the presence of the 50-mer oligonucleotide containing a biotin label
on either the 5` or 3` end. A, sequences of biotin-labeled
oligonucleotides. B, Dda ATPase activity as a function of the
3`-biotin-labeled 50-mer in the presence () or absence (
) of
560 nM streptavidin. The K
determined
for this substrate was 19.8 ± 1.5 µM nt in the
absence of SA and 2.3 ± 0.3 µM nt in the presence
of SA. V
values in the presence and absence of
SA were 3420 ± 80 nM
s
and 3510
± 100 nM
s
, respectively. C, Dda ATPase as a function of the 5`-biotin-labeled 50-mer in
the presence (
) or absence (
) of 560 nM
streptavidin. K
values obtained in the presence
and absence of SA were 24.0 ± 3.0 µM nt and 13.7
± 1.0 µM nt, respectively and V
values in the presence and absence of SA were 3791 ± 147
nM
s
and 3665 ± 52
nM
s
,
respectively.
The overall effect that this change
may have on V and K
is
difficult to evaluate due to the complexity of and . However, for an oligonucleotide, one reasonable
assumption is that k
, the rate constant for
translocation to the end of the DNA lattice, is large relative to k
and k
. This is so
because the lattice is very short, and k
is
length-dependent. Thus, the K
term collapses to , and the term for V
collapses to .
Placement of a block which prevents translocation from the end
of the DNA effectively reduces k to 0, thus the K
term reduces further to in the
presence of the SA block.
Comparison of and enables a clear
prediction that the K will be reduced in the
presence of the SA block. Comparison of V
terms
( and ) indicates that this parameter is not
expected to change in the presence of the block.
Both the
3`-biotinylated and unlabeled 50-mer supported ATPase activity in the
absence of streptavidin (Fig. 7B, Table 2). When
streptavidin was added, the K for ATPase
activity was reduced (Fig. 7B). The data in Fig. 7B provided a K
of 19.8
µM nt for the 3`-biotin 50-mer in the absence of SA and a K
of 2.3 µM nt in the presence of
SA. The large difference observed in the K
values suggests that the Dda is sequestered on the 50-mer,
resulting in an increase in the residence time of Dda on the
oligonucleotide in the presence of SA, as predicted if k
were effectively eliminated. The lack of a significant effect on V
in the presence of the block is also
consistent with the kinetic model ( and ). The
5`-biotin 50-mer stimulated Dda ATPase activity to a similar degree as
did the 3`-biotin 50-mer and the unlabeled 50-mer. The addition of SA
to the 5`-biotin 50-mer had little effect on the Dda ATPase activity (Fig. 7C). The contrasting reduction in K
for the 3`-biotin 50-mer in the presence of SA versus the lack of a similar effect for the 5`-biotin 50-mer
suggests that Dda translocates in a 5` to 3` direction on ssDNA.
We sought to determine whether the increase in ATPase activity on the 3`-biotin 50-mer in the presence of SA was due to a structural effect in the 50-mer oligonucleotide that was removed in the presence of SA. The 23-mer from which the 50-mer was constructed was found to support ATPase activity better than the 50-mer, and thus the 3`-biotin-labeled form of this oligonucleotide was used as a substrate for measuring ATPase activity. The results are shown in Table 3along with results for the 5`-biotin 27-mer. The same trend was observed with these substrates at low DNA concentration, as was seen with the biotin-labeled 50-mers. The effects on Dda ATPase activity observed upon SA binding to the biotinylated oligonucleotides are therefore not due to a structural anomaly in the 50-mer. The low ATPase activity found with the 50-mer is probably due to a sequence effect contained within the 27-mer which also stimulated Dda ATPase activity poorly (Table 3).
Helicases show a preference for unwinding a dsDNA possessing a flanking ssDNA region that serves as an initiation site for the unwinding reaction. The actual substrate may have the ssDNA as 5` to 3` or 3` to 5` with respect to the duplex with the preferred substrate bound determining the polarity of the helicase. The requirement of many helicases for a region of ssDNA implies an association of translocation on ssDNA with unwinding of dsDNA and that the two processes may share a common mechanism. One mechanism for translocation of the E. coli Rep helicase (Wong and Lohman, 1992; Amaratunga and Lohman, 1993) is based on a dimer state for the helicase with two DNA binding sites that alternatively bind to ssDNA or dsDNA in a rolling fashion. The ss/dsDNA junction provides the polarity which ensures unidirectional translocation through dsDNA, but not necessarily ssDNA. Others have proposed that the E. coli Rho helicase (Geiselmann et al., 1993), E. coli UvrD helicase (Matson, 1986), T7 gene 4 helicase (Notarnicola and Richardson, 1994), and T4 41 helicase (Young et al., 1994b) translocate undirectionally along ssRNA or ssDNA.
The question of translocation on ssDNA for the T4 Dda
helicase is addressed here. Previous work (Raney et al., 1994;
Jongeneel et al., 1984; Krell et al., 1979) has
indicated that Dda acts distributively, rapidly dissociating from DNA,
making the directionality of translocation difficult to investigate
with this enzyme. The precise function of Dda in vivo is not
known; however it is believed to be involved in recombination and in
early events in replication (Barry and Alberts, 1994; Gauss et
al., 1994). The activity of Dda is not essential and can be
replaced by the T4 41 helicase in conjunction with the T4 59 protein,
although T4 strains which are both Dda and
59
are not viable (Gauss et al., 1994).
We sought to determine whether the 5` to 3` polarity observed in dsDNA unwinding by Dda (Table 1) is also reflected in the enzyme's translocation on ssDNA. No length dependence for the DNA stimulation of Dda ATPase activity has been demonstrated previously (Jongeneel et al. 1984; Krell et al., 1979). Thus, Dda might operate by a stoichiometric mechanism whereby one molecule of Dda bound near the ss/dsDNA junction could induce local melting of the dsDNA providing a region of ssDNA. A second molecule of Dda could then bind to the newly exposed ssDNA, and this process could lead to directional unwinding of dsDNA without translocation of the helicase.
With varying length oligonucleotides (Table 2), no pattern for
the effect of DNA length on the kinetic parameters, K and V
, was observed. Since DNA sequence
may be important for this helicase, at least with oligonucleotides, we
compared DNA-stimulated ATPase activity in the presence of poly(dT) and
oligo(dT)
(Fig. 4) and observed a clear difference
in Dda ATPase activity. The length-dependent stimulation of helicase
ATPase activity was rationalized by a scheme featuring two species
capable of hydrolyzing ATP. The first includes all forms of E
DNA` where the enzyme is within the substrate lattice;
the second (E
DNA") represents helicase bound at the
ssDNA terminus (Fig. S2). Both species are assigned different
turnover numbers for ATP hydrolysis with their relative concentrations
reflecting the rate of conversion via k
of E
DNA` to E
DNA" by translocation of the
helicase. This linkage explicitly introduces the dependence on ssDNA
length of the ATPase activity.
We then sought data for the
translocation directionality of the helicase on ssDNA by using
substrates containing potential translocation blocks. The T4 32
ssDNA-binding protein had been previously used to inhibit T4 41 ATPase
activity and, presumably, translocation of the helicase (Young et
al., 1994b). Our attempt to apply this method to Dda helicase
indicated that ATPase activity on poly(dT) is not completely inhibited
by 32 and may in fact be stimulated at low 32 concentrations (Fig. 6), consistent with a possible functional interaction
between Dda and T4 32 protein (Formosa et al., 1983). We
therefore sought a different protein block to inhibit Dda
translocation. Some protein blocks do not inhibit Dda unwinding,
suggesting that in these cases, Dda is capable of translocating through
the block (Bedrosian and Bastia, 1991; Yancey-Wrona and Matson, 1992).
Maine and Kodadek(1994) prepared a partial duplex DNA substrate
containing a binding site for GAL4 and reported the reduction in ATPase
activity and DNA unwinding by Dda in the presence of the GAL4DNA
complex. However, this approach does not distinguish between
unidirectional and bidirectional translocation on ssDNA.
Thus, we prepared two ssDNA substrates containing an asymetrically located protein block which would not be displaced by Dda. Two 50-mer oligonucleotides with a biotin label covalently attached on either the 5` or 3` end were constructed, that when bound to SA, provided a translocation block which was unlikely to be displaced (Fig. 7A). A similar substrate was effective in the reconstitution of the T4 polymerase holoenzyme by preventing the T4 45 protein sliding clamp from dissociating from end of the DNA during assembly of the holoenzyme complex (Kaboord and Benkovic, 1993).
In
the absence of SA, these substrates had little or no effect on the
stimulation of the ATPase activity of Dda (compare Fig. 7, B and C, with Table 2). However, in the presence of
SA, a reduction in K for Dda ATPase activity was
observed only with the 3`-biotin labeled 50-mer (Fig. 7B). The 8-fold reduction in K
for this substrate implies that Dda does not dissociate as
rapidly from this DNA strand due to the SA block, consistent with SA
acting to prevent translocation from the 3` end of the oligonucleotide
and thereby effectively eliminating k
in Fig. S2. In contrast, the addition of SA to the
5`-biotin-labeled 50-mer led to only a small increase in the K
for Dda ATPase activity (Fig. 7C).
We interpret these results to indicate
that Dda translocates unidirectionally on ssDNA in a 5` to 3`
direction. The effect of SA at the 3` end is to sequester Dda onto the
DNA, preventing rapid dissociation of the helicase from the end of the
DNA (Fig. 8). One might expect that upon halting helicase
translocation, the V for ATPase activity would
also be reduced, although the results described here indicate that no
reduction has occurred. This observation suggests that enzyme
translocation and ATP hydrolysis may have been decoupled. However, Dda
may rebound from the SA block and continue to catalyze the hydrolysis
of ATP. If this helicase, like all other DNA helicases studied to date,
exists in an oligomeric form, then it should contain multiple DNA
binding sites which can bind DNA in an ordered fashion coupled to ATP
binding and hydrolysis. Thus, while translocation from the end of the
DNA is inhibited, the cycle of DNA binding events proposed to occur
with helicase activity and associated ATP hydrolysis may continue. The
recent observation that the E. coli RuvB helicase binds to DNA
by encircling the DNA (Stasiak et al., 1994) provides an
attractive means for a helicase to remain topologically bound to DNA
while being inhibited from translocating due to the SA block, although
the oligomeric nature of Dda has not been established. We have observed
the same phenomenon with the T4 41 helicase as well; i.e. the
ATPase activity of T4 41 helicase is enhanced by the 3` SA block at low
DNA concentration. (
)
Figure 8:
Diagram depicting Dda helicase
translocation on biotin/streptavidin labeled oligonucleotides. A, 5` to 3` unidirectional translocation leads to rapid
dissociation from the DNA end of the 5`-biotin-labeled oligonucleotide. B, dissociation from the end of the 3`-biotin-labeled
oligonucleotide is blocked in the presence of streptavidin. Dda is
effectively sequestered onto the DNA and K is
lowered.